Antiviral compounds and methods

ABSTRACT

The invention relates to compounds having antiviral and methods utilizing the compounds to treat viral infections.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/602,526, filed May 23, 2017, now allowed, which is a continuation ofU.S. patent application Ser. No. 14/615,616, filed Feb. 6, 2015, whichis a continuation of U.S. patent application Ser. No. 13/553,239, filedJul. 19, 2012, which is a continuation of U.S. application Ser. No.10/562,296, filed Dec. 22, 2005, which is a U.S. national phase of PCTInternational Patent Application No. PCT/AU2004/000866, filed Jun. 26,2004, which claims priority to Australian Application No. 2004902902,filed May 31, 2004, Australian Application No. 2003904692, filed Aug.29, 2003, Australian Application No. 2003903850, filed Jul. 25, 2003,and Australian Application No. 2003903251, filed Jun. 26, 2003, thecontents of each of which are incorporated herein by reference in theirentirety.

FIELD OF INVENTION

The present invention relates to methods for retarding, reducing orotherwise inhibiting viral growth and/or functional activity. Theinvention also relates to compounds and compositions suitable for use inthe methods.

BACKGROUND OF THE INVENTION

Currently, there is a great need for the development of new treatmentsthat are effective against viral infections, particularly against viralinfections which are associated with high morbidity and mortality, andwhich impact on sizable populations. Treatments currently available areinadequate or ineffective in large proportions of infected patients.

For example, in ameliorating AIDS symptoms and prolonging lifeexpectancy, a measure of success has been achieved with drugs targetingthe viral reverse transcriptase and protease enzymes (Miller and Sarver,1997; Mitsuya, 1992; Moore, 1997; and Thomas and Brady, 1997). However,no single treatment method is completely effective against HIVinfection. (Barry et al, 1998; Deeks, 1998; Miles, 1997; Miles, 1998;Moyle et al, 1998; Rachlis and Zarowny, 1998; Veil et al, 1997;Volberding and Deeks, 1998; and Volberdin, 1998).

PCT application PCT/AU99/00872 describes the use of compounds5-(N,N-hexamethylene)-amiloride and 5-(N,N-dimethyl)-amiloride in thetreatment of HIV infection.

Another virus considered to be a significant human pathogen is theHepatitis C virus (HCV). This is a significant human pathogen in termsof both cost to human health and associated economic costs. HCV causeschronic hepatitis and cirrhosis and is the leading indicator for liverreplacement surgery. In 2002 the Centre for Disease Control andPrevention estimated that more than 4 million people were infected inthe USA alone and that approximately 8,000 to 10,000 die as a result ofchronic HCV infection yearly. There is no known cure or vaccine. Moreeffective pharmacological agents are urgently required.

A further well-known family of pathogenic viruses are the Coronaviruses.Coronaviruses (Order Nidovirales, family Coronaviridae, GenusCoronavirus) are enveloped positive-stranded RNA viruses that bud fromthe endoplasmic reticulum-Golgi intermediate compartment or thecis-Golgi network (Fischer, Stegen et al. 1998; Maeda, Maeda et al.1999; Corse and Machamer 2000; Maeda, Repass et al. 2001; Kuo andMasters 2003)

Coronaviruses infect humans and animals and it is thought that therecould be a coronavirus that infects every animal. The two humancoronaviruses, 229E and OC43, are known to be the major causes of thecommon cold and can occasionally cause pneumonia in older adults,neonates, or immunocompromised patients (Peiris, Lai et al. 2003).Animal coronaviruses can cause respiratory, gastrointestinal,neurological, or hepatic diseases in their host (Peiris, Lai et al.2003). Several animal coronavirus are significant veterinary pathogens(Rota, Oberste et al. 2003).

Severe acute respiratory syndrome (SARS) is caused by a newly identifiedvirus. SARS is a respiratory illness that has recently been reported inAsia, North America, and Europe (Peiris, Lai et al. 2003). The causativeagent of SARS was identified as a coronavirus. (Drosten, Gunther et al.2003; Ksiazek, Erdman et al. 2003; Peiris, Lai et al. 2003). The WorldHealth Organization reports that the cumulative number of reportedprobable cases of SARS from 1 Nov. 2002 to the 11 Jul. 2003 is 8,437with 813 deaths, nearly a 10% death rate. It is believed that SARS willnot be eradicated, but will cause seasonal epidemics like the cold orinfluenza viruses (Vogel 2003).

To improve the prospect of treating and preventing viral infections,there is an on-going need to identify molecules capable of inhibitingvarious aspects of the viral life cycle.

It is an object of the present invention to overcome or ameliorate atleast one of the disadvantages of the prior art, or to provide a usefulalternative.

Any discussion of the prior art throughout the specification should inno way be considered as an admission that such prior art is widely knownor forms part of common general knowledge in the field.

SUMMARY OF THE INVENTION

The inventors have surprisingly found that certain compounds that fallunder the classification of substituted acylguanidines have antiviralactivity against viruses from a range of different virus families.Without intending to be bound by any particular theory or mechanism ofaction, and despite current dogma, it appears possible that viralreplication can be retarded by inhibiting or otherwise down-regulatingthe activity of ion channels expressed in the host cell. Thus, thenegative impact of the compounds of the present invention on viralreplication may be mediated by the inhibition or otherwisedown-regulation of a membrane ion channel relied upon by the virus forreplication. This membrane ion channel may be a viral membrane ionchannel (exogenous to the host cell) or a host cell ion channel inducedas a result of viral infection (endogenous to the host cell).

As an example, the compounds of the present invention may inhibit Vpu orp7 function and thereby inhibit the continuation of the respective HIVor HCV life cycle.

The SARS virus encodes an E protein which is shown for the first time,by the present inventors, to act as an ion channel. As similar Eproteins are present in other coronaviruses, the compounds, compositionsand methods of the present invention would have utility in theinhibition and/or treatment of infections by other coronaviruses.

The present invention is concerned with novel antiviral compounds thatfall under the classification of substituted acylguanidines. It does notinclude in its scope the use of compounds 5-(N,N-hexamethylene)amilorideand 5-(N,N-dimethyl)-amiloride for retarding, reducing or otherwiseinhibiting viral growth and/or functional activity of HIV.

Accordingly, a first aspect of the present invention provides anacylguanidine with antiviral activity.

According to a second aspect, the present invention provides anantiviral compound of Formula I

wherein R1-R4 are independently aromatic groups, heteroaromatic groups,

alkylaromatic groups, alkylheteroaromatic groups, alkenylaromaticgroups, alkenylheteroaromatic groups, cycloalkylaromatic groups,cycloalkylheteroaromatic groups, aryloxyalkyl groups, heteroaryloxyalkylgroups, said groups are mono or polycyclic, and are optionallysubstituted with one or more substitutents independently selected fromhydrogen, hydroxy, nitro, halo, amino, substituted amino,alkyl-substituted amino, cycloalkyl-substituted amino, aryl-substitutedamino, C1-6alkyl, C1-6alkyloxy, C3-6cycloalkyl, halo-substitutedC1-6alkyl, halo-substituted C1-6alkyloxy, phenyl, C1-6alkeneyl,C3-6cycloalkeneyl, C1-6alkeneoxy, benzo, aryl, substituted aryl, PrS,

According to a third aspect, the present invention provides an antiviralcompound of Formula I

-   -   or pharmaceutically acceptable salts thereof,    -   wherein,

-   -   R₂, R₃ and R₄ are independently hydrogen,

-   -   and wherein    -   X=hydrogen, hydroxy, nitro, halo, C₁₋₆alkyl, C₁₋₆alkyloxy,        C₃₋₆cycloalkyl, halo-substituted C₁₋₆alkyl, halo-substituted        C₁₋₆alkyloxy, phenyl, C₁₋₆alkeneyl, C₃₋₆cycloalkeneyl,        C₁₋₆alkeneoxy, or benzo;    -   R_(a), R_(b), R_(c), R_(d), R_(e), R_(f), R_(h), R_(k), R_(L),        R_(m), R_(n), R_(o), R_(p) independently=hydrogen, amino, halo,        C₁₋₅alkyl, C₁₋₅alkyloxy, hydroxy, aryl, substituted aryl,        substituted amino, mono or dialkyl-substituted amino,        cycloalkyl-substituted amino, aryl-substituted amino,

-   -   or PrS;    -   R_(g), R_(i) independently=hydrogen, hydroxy, halo, or C₁₋₅        alkyl;    -   R_(j)=hydrogen, amino, halo, C₁₋₅alkyl, C₁₋₅alkyloxy, hydroxy,        aryl, substituted aryl, substituted amino, alkyl-substituted        amino, cycloalkyl-substituted amino, aryl-substituted amino,        PrS,

Preferably, the compounds of the invention include the following:

-   5-(N,N-hexamethylene)amiloride comprising the structure

-   5-(N,N-Dimethyl)amiloride hydrochloride comprising the structure

-   5-(N-methyl-N-isobutyl)amiloride comprising the structure

-   5-(N-ethyl-N-isopropyl)amiloride (herein referred to as EIPA),    comprising the structure

-   N-(3,5-Diamino-6-chloro-pyrazine-2-carbonyl)-N′-phenylguanidine,    comprising the structure

-   N-Benzyl-N′-(3,5-diamino-6-chloro-pyrzine-2-carbonyl)-guanidine,    comprising the structure

-   3-methoxy amiloride comprising the structure comprising the    structure

-   3-methoxy-5-(N,N-Hexamethylene)-amiloride comprising the structure

-   3-hydroxy-5-hexamethyleneimino-amiloride comprising the structure

-   Hexamethyleneimino-6-phenyl-2-pyraxinecarboxamide comprising the    structure

-   N-amidino-3,5-diamino-6-phenyl-2-pyrazinecarboxamide comprising the    structure

-   5-(N,N-hexamethylene)amiloride comprising the structure

-   N-amidino-3-amino-5-phenyl-6-chloro-2-pyrazinecarboxamide comprising    the structure

-   3′4 DichloroBenzamil comprising the structure

-   2′4 DichloroBenzamil HCl comprising the structure

-   5-(N-methyl-N-guanidinocarbonyl-methyl)amiloride comprising the    structure

-   5-(N,N-Diethyl)amiloride hydrochloride comprising the structure

-   5-(N,N-Dimethyl)amiloride hydrochloride comprising the structure

-   5-tert-butylamino-amiloride comprising the structure

-   6-Iodoamiloride comprising the structure

-   Bodipy-FL Amiloride comprising the structure

-   5-(4-fluorophenyl)amiloride comprising the structure

-   1-napthoylguanidine comprising the structure

-   2-napthoylguanidine comprising the structure

-   N-(2-napthoyl)-N′-phenylguanidine comprising the structure

-   N,N′-bis(2-napthoyl)guanidine comprising the structure

-   N,N′-bis(1-napthoyl)guanidine comprising the structure

-   N,N′-bis(2-napthoyl)-N″-phenylguanidine comprising the structure

-   6-methoxy-2-naphthoylguanidine comprising the structure

-   N-Cinnamoyl-N′,N′-dimethylguanidine comprising the structure

-   3-quinolinoylguanidine comprising the structure

-   cinnamoylguanidine comprising the structure

-   4-phenylbenzoylguanidine comprising the structure

-   N-(cinnamoyl)-N′-phenylguanidine comprising the structure

-   (3-phenylpropanoyl)guanidine comprising the structure

-   N,N′-bis-(cinnamoyl)-N″-phenylguanidine comprising the structure

-   N-(3-phenylpropanoyl)-N′-phenylguanidine comprising the structure

-   N,N′-bis(3phenylpropanoyl)-N″-phenylguanidine comprising the    structure

-   trans-3-furanacryoylguanidine comprising the structure

-   N-(6-Hydroxy-2-napthoyl)-N′-phenylguanidine comprising the structure

-   (4-Phenoxybenzoyl)guanidine comprising the structure

-   N,N′-Bis(amidino)napthalene-2,6-dicarboxamide comprising the    structure

-   6-bromo-2-napthoylguanidine comprising the structure

-   1-bromo-2-napthoylguanidine comprising the structure

-   2-(2-napthyl)acetoylguanidine comprising the structure

-   N″-Cinnamoyl-N,N′-diphenylguanidine comprising the structure

-   (Phenylacetyl)guanidine comprising the structure

-   N,N-Bis(3-phenylpropanoyl)guanidine comprising the structure

-   Benzoylguanidine comprising the structure

-   (4-Chlorophenoxy-acetyl)guanidine comprising the structure

-   N-Benzoyl-N′-cinnamoylguanidine comprising the structure

-   [(E)-3-(4-Dimethylaminophenyl)-2-methylacryloyl]guanidine comprising    the structure

-   (4-Chlorocinnamoyl)guanidine comprising the structure

-   (4-Bromocinnamoyl)guanidine comprising the structure

-   (4-Methoxycinnamoyl)guanidine comprising the structure

-   (5-Phenyl-penta-2,4-dienoyl)guanidine comprising the structure

-   (3-Bromocinnamoyl)guanidine comprising the structure

-   (3-Methoxycinnamoyl)guanidine comprising the structure

-   (3-Chlorocinnamoyl)guanidine comprising the structure

-   (2-Chlorocinnamoyl)guanidine comprising the structure

-   (2-Bromocinnamoyl)guanidine comprising the structure

-   (2-Methoxycinnamoyl)guanidine comprising the structure

-   (trans-2-Phenylcyclopropanecarbonyl)guanidine comprising the    structure

-   [3-(3-Pyridyl)acryloyl]guanidine comprising the structure

-   (4-Hydroxycinnamoyl)guanidine comprising the structure

-   (Quinoline-2-carbonyl)guanidine comprising the structure

-   (4-Nitrocinnamoyl)guanidine comprising the structure

-   (3-Nitrocinnamoyl)guanidine comprising the structure

-   (2-Nitrocinnamoyl)guanidine comprising the structure

-   (α-Methylcinnamoyl)guanidine comprising the structure

-   trans-3-(1-napthyl)acryloylguanidine comprising the structure

-   4-phenylcinnamoylguanidine comprising the structure

-   3-(trifluoromethyl)cinnamoylguanidine comprising the structure

-   3-methylcinnamoylguanidine comprising the structure

-   4-(trifluoromethyl)cinnamoylguanidine comprising the structure

-   2-methylcinnamoylguanidine comprising the structure

-   2-(trifluoromethyl)cinnamoylguanidine comprising the structure

-   4-methylcinnamoylguanidine comprising the structure

-   4-isopropylcinnamoylguanidine comprising the structure

-   3-fluorocinnamoylguanidine comprising the structure

-   2-fluorocinnamoylguanidine comprising the structure

-   4-fluorocinnamoylguanidine comprising the structure

-   3,4-dichlorocinnamoylguanidine comprising the structure

-   2,4-dichlorocinnamolyguanidine comprising the structure

-   2,6-dichlorocinnamoylguanidine comprising the structure

-   4-ethoxycinnamoylguanidine comprising the structure

-   3,4-(methylenedioxy)cinnamoylguanidine comprising the structure

-   3-(2-napthyl)acryloylguanidine comprising the structure

-   4-t-butylcinnamoylguanidine comprising the structure

-   3,4,5-trimethoxycinnamoylguanidine comprising the structure

-   2-(1-napthyl)acetoylguanidine comprising the structure

-   2,5-dimethylcinnamoylguanidine comprising the structure

-   2,3-difluorocinnamoylguanidine comprising the structure

-   3-phenylcinnamoylguanidine comprising the structure

-   3-(trans-hept-1-en-1-yl)cinnamoylguanidine comprising the structure

-   2-ethylcinnamoylguanidine comprising the structure

-   2-chloro-6-fluorocinnamoylguanidine comprising the structure

-   3-t-butylcinnamoylguanidine comprising the structure

-   3,4-difluorocinnamoylguanidine comprising the structure

-   5-bromo-2-fluorocinnamoylguanidine comprising the structure

-   3-(trifluoromethoxy)cinnamoylguanidine comprising the structure

-   2-ethoxycinnamoylguanidine comprising the structure

-   2-t-butylcinnamoylguanidine comprising the structure

-   3-(cyclohex-1-en-1-yl)cinnamoylguanidine comprising the structure

-   cinnamoylguanidine hydrochloride comprising the structure

-   2,3,5,6,-tetramethylcinnamoylguanidine comprising the structure (Bit    134)

-   2-cyclohexylcinnamoylguanidine comprising the structure

-   5-bromo-2-methoxycinnamoylguanidine comprising the structure

-   2,3-dimethylcinnamoylguanidine comprising the structure

-   3-ethoxycinnamoylguanidine comprising the structure

-   3-isopropylcinnamoylguanidine hydrochloride comprising the structure

-   2-phenylcinnamoylguanidine comprising the structure

-   2-(cyclohex-1-en-1yl)cinnamoylguanidine comprising the structure

-   2,4,6-trimethylcinnamoylguanidine comprising the structure

-   (5-Phenyl-penta-2,4-dienoyl)guanidine comprising the structure

-   5-(3′-bromophenyl)penta-2,4-dienoylguanidine comprising the    structure

-   5-(2′-bromophenyl)penta-2,4-dienoylguanidine comprising the    structure

-   Furanacryloyl comprising the structure

Preferably, the compounds of the invention are capable of reducing,retarding or otherwise inhibiting viral growth and/or replication.

Preferably, the antiviral activity of the compounds of the invention isagainst viruses such as those belonging to the Lentivirus family, andthe Coronovirus family family of viruses. For example, the compounds ofthe invention exhibit antiviral activity against viruses such as HumanImmunodeficiency Virus (HIV), Severe Acute Respiratory Syndrome virus(SARS), Mouse Hepatitis virus ( ), and Hepatitis C virus (HCV).

According to a fourth aspect of the present invention, there is provideda pharmaceutical composition comprising an antiviral compound accordingto any one of the first, second or third aspects, and optionally one ormore pharmaceutical acceptable carriers or derivatives, wherein saidcompound is capable of reducing, retarding or otherwise inhibiting viralgrowth and/or replication.

Preferably, the antiviral activity of the compounds of the invention isagainst viruses such as those belonging to the Lentivirus family, andthe Coronovirus family of viruses. For example, the compounds of theinvention exhibit antiviral activity against viruses such as HumanImmunodeficiency Virus (HIV), Severe Acute Respiratory Syndrome virus(SARS), Human Coronavirus 229E, Human Coronavirus OC43, Mouse Hepatitisvirus (MHV), Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus(PRCV), Hepatitis C virus (HCV) and Equine Arteritis Virus (EAV).

Other Coronaviruses which can be inhibited or their infections treatedby the compounds of the invention are those listed in Table 1.

The compositions of the invention may further comprise one or more knownantiviral compounds or molecules.

According to a fifth aspect, there is provided a method for reducing,retarding or otherwise inhibiting growth and/or replication of a viruscomprising contacting a cell infected with said virus or exposed to saidvirus with a compound according to any one of the first, second or thirdaspects.

Preferably, the virus is from the Lentivirus family, or the Coronavirusfamily. More preferably, the virus is Human Immunodeficiency Virus(HIV), Severe Respiratory Syndrome virus (SARS), Human Coronavirus 229E,Human Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus(BCV), Porcine Respiratory Coronavirus (PRCV), Mouse Hepatitis virus(MHV), Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Mostpreferably, the virus is HIV-1, HIV-2, the SARS virus, Coronaviruse229E, Coronavirus OC43, PRCV, BCV, HCV, or EAV.

Other Coronaviruses which can be inhibited or their infections treatedby the compounds of the invention are those listed in Table 1.

According to a sixth aspect, there is provided a method for preventingthe infection of a cell exposed to a virus comprising contacting saidcell with a compound according to any one of the first, second or thirdaspects.

Preferably, the virus is from the Lentivirus family, or the Coronavirusfamily. More preferably, the virus is Human Immunodeficiency Virus(HIV), Severe Respiratory Syndrome virus (SARS), Human Coronavirus 229E,Human Coronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus(BCV), Porcine Respiratory Coronavirus (PRCV), Mouse Hepatitis virus(MHV), Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Mostpreferably, the virus is HIV-1, HIV-2, the SARS virus, Coronaviruse229E, Coronavirus OC43, PRCV, BCV, HCV, EAV.

Other Coronaviruses which can be inhibited or their infections treatedby the compounds of the invention are those listed in Table 1.

According to a seventh aspect of the invention, there is provided amethod for the therapeutic or prophylactic treatment of a subjectinfected with or exposed to a virus, comprising the administration of acompound according to any one of the first, second or third aspects, toa subject in need of said treatment.

Preferably, infection with a virus or exposure to a virus occurs withviruses belonging to the Lentivirus family, or the Coronovirus family.More preferably, infection or exposure occurs with HIV, SARS, HumanCoronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MHV),Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV),Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Mostpreferably, infection or exposure occurs with HIV-1, HIV-2, SARS, HumanCoronavirus 229E, Human Coronavirus OC43, Hepatitis C virus (HCV), orEquine Arteritis Virus (EAV).

Other Coronaviruses which can be inhibited or their infections treatedby the compounds of the invention are those listed in Table 1.

The subject of the viral inhibition is generally a mammal such as butnot limited to human, primate, livestock animal (e.g. sheep, cow, horse,donkey, pig), companion animal (e.g. dog, cat), laboratory test animal(e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animal(e.g. fox, deer). Preferably, the subject is a primate, or horse. Mostpreferably, the subject is a human.

According to a eighth aspect, there is provided a method of downregulating a membrane ion channel functional activity in a cell infectedwith a virus, comprising contacting said cell with a compound accordingto any one of the first, second or third aspects.

The membrane ion channel may be endogenous to the cell or exogenous tothe cell.

Preferably, the membrane ion channel of which functional activity isdown regulated is that which Lentiviruses, and Coronaviruses utilise formediating viral replication and include, for example, the HIV membraneion channel Vpu, the HCV membrane ion channel P7, the Coronavirus Eprotein membrane ion channel, and the SARS E protein membrane ionchannel.

Preferably, infection with a virus or exposure to a virus occurs withviruses belonging to the Lentivirus family, or the Coronovirus family.More preferably, infection or exposure occurs with HIV, SARS, HumanCoronavirus 229E, Human Coronavirus OC43, Mouse Hepatitis virus (MHV),Bovine Coronavirus (BCV), Porcine Respiratory Coronavirus (PRCV),Hepatitis C virus (HCV), or Equine Arteritis Virus (EAV). Mostpreferably, infection or exposure occurs with HIV-1, HIV-2, SARS, HumanCoronavirus 229E, Human Coronavirus OC43, Hepatitis C virus (HCV), orEquine Arteritis Virus (EAV).

According to an ninth aspect of the present invention, there is provideda method of reducing, retarding or otherwise inhibiting growth and/orreplication of a virus that has infected a cell, said method comprisingcontacting said infected cell with a compound according to any one ofthe first, second or third aspects, wherein said compound down regulatesfunctional activity of a membrane ion channel derived from said virusand expressed in said infected cell.

Preferably, infection occurs with a virus belonging to the Lentivirusfamily, or the Coronovirus family. More preferably, infection orexposure occurs with HIV, SARS, Human Coronavirus 229E, HumanCoronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV),Porcine Respiratory Coronavirus (PRCV), Hepatitis C virus (HCV), orEquine Arteritis Virus (EAV). Most preferably, infection or exposureoccurs with HIV-1, HIV-2, SARS, Human Coronavirus 229E, HumanCoronavirus OC43, Hepatitis C virus (HCV), or Equine Arteritis Virus(EAV).

Other Coronaviruses which can be inhibited or their infections treatedby the compounds of the invention are those listed in Table 1.

Preferably, the membrane ion channel of which functional activity isdown regulated is that which Lentiviruses, and Coronaviruses utilise formediating viral replication and include, for example, the HIV membraneion channel Vpu, the HCV membrane ion channel P7, and the Coronavirus Eprotein membrane ion channel.

According to an tenth aspect, the present invention provides a method ofreducing, retarding or otherwise inhibiting growth and/or replication ofa virus that has infected a cell in a mammal, said method comprisingadministering to said mammal a compound according to any one of thefirst, second or third aspects, or a pharmaceutical compositionaccording to the fourth aspect, wherein said compound or saidcomposition down regulates functional activity of a membrane ion channelexpressed in said infected cell.

Preferably, infection occurs with a virus belonging to the Lentivirusfamily, or the Coronovirus family. More preferably, infection orexposure occurs with HIV, SARS, Human Coronavirus 229E, HumanCoronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV),Porcine Respiratory Coronavirus (PRCV), Hepatitis C virus (HCV), orEquine Arteritis Virus (EAV). Most preferably, infection or exposureoccurs with HIV-1, HIV-2, SARS, Human Coronavirus 229E, HumanCoronavirus OC43, Hepatitis C virus (HCV), or Equine Arteritis Virus(EAV).

Other Coronaviruses which can be inhibited or their infections treatedby the compounds of the invention are those listed in Table 1.

Preferably, the membrane ion channel of which functional activity isdown regulated is that which Lentiviruses, and Coronaviruses utilise formediating viral replication and include, for example, the HIV membraneion channel Vpu, the HCV membrane ion channel P7, and the Coronavirus Eprotein membrane ion channel.

The subject of the viral inhibition is generally a mammal such as butnot limited to human, primate, livestock animal (e.g. sheep, cow, horse,donkey, pig), companion animal (e.g. dog, cat), laboratory test animal(e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animal(e.g. fox, deer). Preferably, the subject is a primate, or horse. Mostpreferably, the subject is a human.

According to a eleventh aspect, the present invention provides a methodfor the therapeutic or prophylactic treatment of a subject infected withor exposed to a virus comprising administering to said subject acompound according to any one of the first, second or third aspects, ora pharmaceutical composition according to the fourth aspect, whereinsaid compound or said composition down-regulates functional activity ofa membrane ion channel derived from said virus.

Preferably, infection occurs with a virus belonging to the Lentivirusfamily, or the Coronovirus family of viruses. More preferably, infectionor exposure occurs with HIV, SARS, Human Coronavirus 229E, HumanCoronavirus OC43, Mouse Hepatitis virus (MHV), Bovine Coronavirus (BCV),Porcine Respiratory Coronavirus (PRCV), Hepatitis C virus (HCV), orEquine Arteritis Virus (EAV). Most preferably, infection or exposureoccurs with HIV-1, HIV-2, SARS, Human Coronavirus 229E, HumanCoronavirus OC43, Hepatitis C virus (HCV), or Equine Arteritis Virus(EAV).

Other Coronaviruses which can be inhibited or their infections treatedby the compounds of the invention are those listed in Table 1.

Preferably, the membrane ion channel of which functional activity isdown regulated is that which Lentiviruses, and Coronaviruses utilise formediating viral replication and include, for example, the HIV membraneion channel Vpu, the HCV membrane ion channel P7, and the Coronavirus Eprotein membrane ion channel.

The subject of the viral inhibition is generally a mammal such as butnot limited to human, primate, livestock animal (e.g. sheep, cow, horse,donkey, pig), companion animal (e.g. dog, cat), laboratory test animal(e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animal(e.g. fox, deer). Preferably, the subject is a primate, or horse. Mostpreferably, the subject is a human.

According to a twelfth aspect, the invention provides an antiviralcompound selected from the group consisting of:

-   -   N-(3,5-Diamino-6-chloro-pyrazine-2-carbonyl)-N′-phenyl-guanidine,    -   N-Benzyl-N′-(3,5-diamino-6-chloro-pyrazine-2-carbonyl)-guanidine,    -   3′4 DichloroBenzamil,    -   2′4 DichloroBenzamil,    -   5-(N-methyl-N-guanidinocarbonyl-methyl)amiloride,    -   5-(N-Methyl-N-isobutyl)amiloride,    -   5-(N-Ethyl-N-isopropyl)amiloride,    -   5-(N,N-Dimethyl)amiloride hydrochloride,    -   5-(N,N-hexamethylene)amiloride,    -   5-(N,N-Diethyl)amiloride hydrochloride,    -   6-Iodoamiloride,    -   Bodipy-FL amiloride,    -   3-hydroxy-5-hexamethyleneimino-amiloride,    -   5-(4-fluorophenyl)amiloride,    -   5-tert-butylamino-amiloride,    -   N-amidino-3-amino-5-phenyl-6-chloro-2-pyrazinecarboxamide,    -   3-methoxy-5-(N,N-Hexamethylene)-amiloride,    -   3-methoxy-amiloride,    -   hexamethyleneimino-6-phenyl-2-pyrazinecarboximide,    -   N-amidino-3,5-diamino-6-phenyl-2-pyrazinecarboxamide,    -   1-naphthoylguanidine,    -   2-naphthoylguanidine,    -   N-(2-naphthoyl)-N′-phenyl-guanidine,    -   N,N′-bis(2-naphthoyl)guanidine,    -   N,N′-bis(1-naphthoyl)guanidine,    -   N,N′-bis(2-naphthoyl)-N″-phenyl-guanidine,    -   6-methoxy-2-naphthoylguanidine,    -   3-quinolinoylguanidine,    -   cinnamoylguanidine,    -   4-phenylbenzoylguanidine,    -   N-(cinnamoyl)-N′-phenyl-guanidine,    -   (3-phenylpropanoyl)guanidine,    -   N,N′-bis-(cinnamoyl)-N″-phenyl-guanidine,    -   N-(3-phenylpropanoyl)-N′-phenyl-guanidine,    -   N,N′-bis(3phenylpropanoyl)-N″-phenyl-guanidine,    -   trans-3-furanacryoylguanidine,    -   N-(6-Hydroxy-2-naphthoyl)-N′-phenyl-guanidine,    -   (4-Phenoxybenzoyl)guanidine,    -   N,N′-Bis(amidino)napthalene-2,6-dicarboxamide,    -   N″-Cinnamoyl-N,N′-diphenylguanidine,    -   (Phenylacetyl)guanidine,    -   N,N′-Bis(3-phenylpropanoyl)guanidine,    -   benzoylguanidine,    -   (4-Chlorophenoxy-acetyl)guanidine,    -   N-benzoyl-N′-cinnamoylguanidine,    -   [(E)-3-(4-Dimethylaminophenyl)-2-methylacryloyl]guanidine,    -   (4-Chlorocinnamoyl)guanidine,    -   (4-Bromocinnamoyl)guanidine,    -   (4-Methoxycinnamoyl)guanidine,    -   (5-Phenyl-penta-2,4-dienoyl)guanidine,    -   (3-Bromocinnamoyl)guanidine,    -   (3-Methoxycinnamoyl)guanidine,    -   (3-Chlorocinnamoyl)guanidine,    -   (2-Chlorocinnamoyl)guanidine,    -   (2-Bromocinnamoyl)guanidine,    -   (2-Methoxycinnamoyl)guanidine,    -   (trans-2-Phenylcyclopropanecarbonyl)guanidine,    -   [3-(3-Pyridyl)acryloyl]guanidine,    -   (4-Hydroxycinnamoyl)guanidine,    -   (Quinoline-2-carbonyl)guanidine,        or pharmaceutically acceptable salts thereof.

According to a thirteenth aspect, the present invention provides apharmaceutical composition comprising a compound according to thetwelfth aspect, and optionally one or more pharmaceutical acceptablecarriers or derivatives.

Preferably, the pharmaceutical composition may further comprise one ormore known antiviral compounds or molecules.

Unless the context clearly requires otherwise, throughout thedescription and the claims, the words ‘comprise’, ‘comprising’, and thelike are to be construed in an inclusive sense as opposed to anexclusive or exhaustive sense; that is to say, in the sense of“including, but not limited to”.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1C is a schematic representation of plasmids used for expressionof Vpu in E. coli. 1A. The amino acid sequence (<400>1) (SEQ ID NO: 1)encoded by the vpu open reading frame (ORF) generated by PCR from anHIV-1 strain HXB2 oDNA clone. The vpu ORF was cloned in-frame at the 3′end of the GST gene in p2GBX to generate p2GEXVpu (1B). It wassubsequently cloned into pPL451 to produce the plasmid pPL+Vpu (1C).

FIG. 2A-2B is a photographic representation of the expression andpurification of Vpu in E. coli. 2A. Western blotting after SDS-PAGE wasused to detect expressed Vpu in E. coli extracts. Lanes 1-4 containsamples, at various stages of purity, of Vpu expressed from p2GEXVpu:lane 1, GST-Vpu flasiors protein isolated by glutathione-agaroseaffinity chromatography; lane 2, Vpu liberated from the fusion proteinby treatment with thrombin; lane 35 Vpu purified by HPLC anion exchangechromatography; lane 4, Vpu after passage through the immunoaffinitycolumn. Lanes 5 and 6, membrane vesicles prepared from 42′C inducedcells containing pPL+Vpu or pPL451, respectively. 2B. Silver stainedSDS-PAGE gel: lane 1, Vpu purified by HPLC anion exchangechromatography; lane 2, Vpu after passage through the immunoaffinitycolumn.

FIG. 3A-3B is a graphical representation of ion channel activityobserved after exposure of lipid bilayers to aliquots containingpurified Vpu. In 3A and 3B, the CIS chamber contained 500 mM NaCl andthe TRANS chamber contained 50 mM NaCl; both solutions were buffered atpH 6.0 with 10 mM MES. 3B shows a current virus voltage curve generatedfrom data similar to that shown in A.

FIG. 4A-4C is a photographic representation of bacterial cross-feedingassays. For all plates, the Met⁻, Pro⁻ auxotrophic strain was used toseed a soft agar overlay. Plates 4A and 4B contain minimal drop-outmedium minus proline; in plate 4C the medium was minus methionine. Tocontrol for viability of the cells in the background lawn, the discslabelled P and M contained added proline or methionine, respectively.The discs labelled C and V were inoculated with Met+, Prof E. coli cellscontaining the plasmids pPL451, or pPL+Vpu, respectively. Plates wereincubated at 37° C. (4A and 4C) or 30° C. (4B) for two days andphotographed above a black background with peripheral illumination froma fluorescent light located below the plate. The images were recorded ona Novaline video gel documentation system. Light halos around the discslabelled P or M on all plates and around the disc labelled V on plate Aindicate growth of the background lawn strain.

FIG. 5 is a graphical representation of the screening of drugs forpotential Vpu channel blockers. The photograph shows a section of aminimal medium-lacking adenine-agarose plate onto which a lawn ofXL-1-blue E. coli cells containing the Vpu expression plasmid pPLVpu hasbeen seeded. Numbers 6-11 are located at the sites of application ofvarious drugs being tested, which were applied in 3 μl drops and allowedto soak into the agarose. The plate was then incubated at 37° C. for 48hr prior to being photographed. The background grey shade corresponds toareas of no bacterial growth. The bright circular area around “10”represents bacterial cell growth as a result of application of adenineat that location (positive control). The smaller halo of bacterialgrowth around “9” is due to the application of5-(N,N-hexamethylene)-amiloride at that location.

FIG. 6A-6B. SARS E protein ion channel activity observed in NaClsolutions after exposure of lipid bilayer to 3-10 mg of E protein. 6A.The closed state is shown as solid line, openings are derivations fromthe line. Scale bar is 300 ms and 5 pA. The CIS chamber contained 50 mMNaCl in 5 mM HEPES buffer pH 7.2, the TRANS chamber contained 500 mMNaCl in 5 mM HEPES buffer pH 7.2. The CIS chamber was earthed and theTRANS chamber was held at various potentials between −100 to +100 mV.6B. Largest single opening events of a single channel.

FIG. 7A-7B. SARS E protein ion channel activity observed in NaClsolutions after exposure of lipid bilayer to 3-10 mg of E protein. 7A.The closed state is shown as solid line, openings are derivations fromthe line. Scale bar is 300 ms and 5 pA. The CIS chamber contained 50 mMNaCl in 5 mM HEPES buffer pH 7.2. the TRANS chamber contained 500 mMNaCl in 5 mM HEPES buffer pH 7.2. The CIS chamber was earthed and theTRANS chamber was held at various potentials between −100 to +100 mV.7B. Largest single opening events of a single channel.

FIG. 8A-8C. Cinnamoylguanidine (Bit036) inhibits SARS E protein ionchannel activity in NaCl solution. 8A. Representative currents atholding potential of −40 mV. Scale bar is 300 mS and 5 pA. E protein ionchannel activity and E protein channel activity after the addition of100 .mu.M Bit036. 8B. All points histogram at holding potential of −40mV. E protein ion channel activity before and after the addition of 100mM Bit036. 8C. Average current (pA), before formation of E protein ionchannel, E protein ion channel activity and after addition of 100 .mu.pMBit036.

FIG. 9: 229E protein ion channel activity in lipid bilayers in KClsolutions.

FIG. 10A-10B: Part 10A shows raw currents generated by the 229E-Eprotein ion channel in a planar lipid bilayer. The top trace showscurrent activity prior to drug addition and the lower trace shows theeffect of addition of 100 μM cinnamoylguanidine on channel activity.Part 10B is a graphical representation of the average current flowingacross the bilayer (in arbitrary units), before and after addition ofcinnamoylguanidine.

FIG. 11: MHV E protein Ion channel activity in lipid bilayars NaClsolutions.

FIG. 12A-B: Part 12A shows raw currents generated by the MHV-E proteinion channel in a planar lipid bilayer. The top trace shows currentactivity prior to drug addition and the lower trace shows the effect ofaddition of 100 μM cinnamoylguanidine on channel activity. Part 12B is agraphical representation of the average current flowing across thebilayer (in arbitrary units), before and after addition ofcinnamoylguanidine.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the surprising determinationthat certain compounds that fall under the classification of substitutedacylguanidines have antiviral activity against viruses from a range ofdifferent virus families. Without intending to be bound by anyparticular theory or mechanism of action, the negative impact of thecompounds of the present invention on viral replication may be mediatedby the inhibition or otherwise down-regulation of a membrane ion channelrelied upon by the virus for replication. This membrane ion channel maybe a viral membrane ion channel (exogenous to the host cell) or a hostcell ion channel induced as a result of viral infection (endogenous tothe host cell).

As an example, the compounds of the present invention may inhibit Vpu orp7 function and thereby inhibit the continuation of the respective HIVor HCV life cycle.

The SARS virus encodes an E protein which is shown for the first time,by the present inventors, to act as an ion channel. As similar Eproteins are present in other coronaviruses, the compounds, compositionsand methods of the present invention would have utility in theinhibition and/or treatment of infections by other coronaviruses.

While the present invention is concerned with novel antiviral compoundsfalling under the classification of substituted acylguanidines, it doesnot include in its scope the use of compounds5-(N,N-hexamethylene)amiloride and 5-(N,N-dimethyl)-amiloride forretarding, reducing or otherwise inhibiting viral growth and/orfunctional activity of HIV.

It will be understood by those skilled in the art that the compounds ofthe invention may be administered in the form of a composition orformulation comprising pharmaceutically acceptable carriers andexcipients.

The pharmaceutical compositions of the invention may further compriseone or more known antiviral compounds or molecules. Preferably, theknown anti-viral compounds are selected from the group consisting ofVidarabine, Acyclovir, Ganciclovir, Valganciclovir, Valacyclovir,Cidofovir, Famciclovir, Ribavirin, Amantadine, Rimantadine, Interferon,Oseltamivir, Palivizumab, Rimantadine, Zanamivir, nucleoside-analogreverse transcriptase inhibitors (NRTI) such as Zidovudine, Didanosine,Zalcitabine, Stavudine, Lamivudine and Abacavir, non-nucleoside reversetranscriptase inhibitors (NNRTI) such as Nevirapine, Delavirdine andEfavirenz, protease inhibitors such as Saquinavir, Ritonavir, Indinavir,Nelfinavir, Amprenavir, and other known antiviral compounds andpreparations. Known anti-viral compounds or molecules may in some casesact synergistically with the anti-viral compounds of the invention.

TABLE 1 Known coronavirus isolates Group 1 species  Canine coronavirus  Canine enteric coronavirus (strain INSAVC-1)   Canine entericcoronavirus (strain K378)  Feline coronavirus   Feline entericcoronavirus (strain 79-1683)   Feline infectious peritonitis virus(FIPV)  Human coronavirus 229E  Porcine epidemic diarrhea virus  Porcine epidemic diarrhea virus (strain Br1/87)   Porcine epidemicdiarrhea virus (strain CV777)  Transmissible gastroenteritis virus  Porcine respiratory coronavirus   Porcine transmissiblegastroenteritis coronavirus (STRAIN   FS772/70)   Porcine transmissiblegastroenteritis coronavirus (strain Miller)   Porcine transmissiblegastroenteritis coronavirus (strain   Neb72-RT)   Porcine transmissiblegastroenteritis coronavirus (STRAIN   PURDUE) Group 2 species  Bovinecoronavirus   Bovine coronavirus (STRAIN F15)   Bovine coronavirus(strain G95)   Bovine coronavirus (STRAIN L9)   Bovine coronavirus(strain LSU-94LSS-051)   Bovine coronavirus (STRAIN LY-138)   Bovinecoronavirus (STRAIN MEBUS)   Bovine coronavirus (strain OK-0514-3)  Bovine coronavirus (strain Ontario)   Bovine coronavirus (STRAINQUEBEC)   Bovine coronavirus (STRAIN VACCINE)   Bovine entericcoronavirus (strain 98TXSF-110-ENT)  Canine respiratory coronavirus Chicken enteric coronavirus  Human coronavirus OC43  Murine hepatitisvirus   Murine coronavirus (strain DVIM)   Murine hepatitis virus(strain A59)   Murine hepatitis virus (strain JHM)   Murine hepatitisvirus (strain S)   Murine hepatitis virus strain 1   Murine hepatitisvirus strain 2   Murine hepatitis virus strain 3   Murine hepatitisvirus strain 4   Murine hepatitis virus strain ML-11  Porcinehemagglutinating encephalomyelitis virus   Porcine hemagglutinatingencephalomyelitis virus (strain 67N)   Porcine hemagglutinatingencephalomyelitis virus (strain IAF-   404)  Puffinosis virus  Ratcoronavirus   Rat coronavirus (strain 681)   Rat coronavirus (strain NJ)  Rat sialodacryoadenitis coronavirus Group 3 species  Turkeycoronavirus   Turkey coronavirus (strain Indiana)   Turkey coronavirus(strain Minnesota)   Turkey coronavirus (strain NC95)  Avian infectiousbronchitis virus   Avian infectious bronchitis virus (STRAIN 6/82)  Avian infectious bronchitis virus (strain Arkansas 99)   Avianinfectious bronchitis virus (strain Beaudette CK)   Avian infectiousbronchitis virus (strain Beaudette M42)   Avian infectious bronchitisvirus (strain Beaudette US)   Avian infectious bronchitis virus (strainBeaudette)   Avian infectious bronchitis virus (strain D1466)   Avianinfectious bronchitis virus (strain D274)   Avian infectious bronchitisvirus (strain D3896)   Avian infectious bronchitis virus (strain D41)  Avian infectious bronchitis virus (strain DE072)   Avian infectiousbronchitis virus (strain GRAY)   Avian infectious bronchitis virus(strain H120)   Avian infectious bronchitis virus (strain H52)   Avianinfectious bronchitis virus (strain KB8523)   Avian infectiousbronchitis virus (strain M41)   Avian infectious bronchitis virus(strain PORTUGAL/322/82)   Avian infectious bronchitis virus (strainSAIB20)   Avian infectious bronchitis virus (strain UK/123/82)   Avianinfectious bronchitis virus (strain UK/142/86)   Avian infectiousbronchitis virus (strain UK/167/84)   Avian infectious bronchitis virus(strain UK/183/66)   Avian infectious bronchitis virus (strain UK/68/84)  Avian infectious bronchitis virus (strain V18/91)   Avian infectiousbronchitis virus (strain Vic S)   Avian infectious laryngotracheitisvirus Preliminary Group 4 species  SARS coronavirus   SARS coronavirusBeijing ZY-2003   SARS coronavirus BJ01   SARS coronavirus BJ02   SARScoronavirus BJ03   SARS coronavirus BJ04   SARS coronavirus CUHK-Su10  SARS coronavirus CUHK-W1   SARS coronavirus Frankfurt 1   SARScoronavirus GZ01   SARS coronavirus HKU-39849   SARS coronavirus HongKong ZY-2003   SARS coronavirus Hong Kong/03/2003   SARS coronavirus HSR1   SARS coronavirus Sin2500   SARS coronavirus Sin2677   SARScoronavirus Sin2679   SARS coronavirus Sin2748   SARS coronavirusSin2774   SARS coronavirus Taiwan   SARS coronavirus Taiwan JC-2003  SARS coronavirus Taiwan TC1   SARS coronavirus Taiwan TC2   SARScoronavirus Tor2   SARS coronavirus TW1   SARS coronavirus TWC   SARScoronavirus Urbani   SARS coronavirus Vietnam   SARS coronavirus ZJ-HZ01  SARS coronavirus ZJ01  unclassified coronaviruses   Bovine respiratorycoronavirus (strain 98TXSF-110-LUN)  Human enteric coronavirus 4408 Enteric coronavirus  Equine coronavirus   Equine coronavirus NC99

The present observations and findings now permit the use of agents suchas certain substituted acylguanidines, as anti-viral agents for thetherapy and prophylaxis of viral conditions caused by different viruses.The methods and compositions of the present invention may beparticularly effective against viruses which rely on ion channelformation for their replication, however it will be understood that thisis not the only mechanism relied on by viruses for replication and thatthe compounds and methods of the present invention are not limited toagents which exert their action by retarding or inhibiting the functionof ion channels.

Reference to “membrane ion channel” should be understood as a referenceto a structure which transports ions across a membrane. The presentinvention extends to ion channels which may function by means such aspassive, osmotic, active or exchange transport. The ion channel may beformed by intracellular or extracellular means. For example, the ionchannel may be an ion channel which is naturally formed by a cell tofacilitate its normal functioning. Alternatively, the ion channel may beformed by extracellular means. Extracellular means would include, forexample, the formation of ion channels due to introduced chemicals,drugs or other agents such as ionophores or due to the functionalactivity of viral proteins encoded by a virus which has entered a cell.

The ion channels which are the subject of certain embodiments of thepresent invention facilitate the transport of ions across membranes.Said membrane may be any membrane and is not limited to the outer cellwall plasma membrane. Accordingly, “membrane” as used herein encompassesthe membrane surrounding any cellular organelle, such as the Golgiapparatus and endoplasmic reticulum, the outer cell membrane, themembrane surrounding any foreign antigen which is located within thecell (for example, a viral envelope) or the membrane of a foreignorganism which is located extracellularly. The membrane is typically,but not necessarily, composed of a fluid lipid bilayer. The subject ionchannel may be of any structure. For example, the Vpu ion channel isformed by Vpu which is an integral membrane protein encoded by HIV-1which associates with, for example, the Golgi and endoplasmic reticulummembranes of infected cells. Reference hereinafter to “Vpu ion channels”is a reference to all related ion channels for example P7 HCV and M2 ofinfluenza and the like.

Reference to “HIV”, “SARS”, “Coronavirus” or “HCV” should be understoodas a reference to any HIV, SARS, Coronavirus or HCV virus strain andincluding homologues and mutants.

Reference to the “functional activity” of an ion channel should beunderstood as a reference to any one or more of the functions which anion channel performs or is involved in. For example, the Vpu proteinencoded ion channel, in addition to facilitating the transportation ofNa⁺, K⁺, Cl⁻ and P0₄ ³⁻, also plays a role in the degradation of the CD4molecule in the endoplasmic reticulum. Without wishing to be bound by aparticular theory, the Vpu protein encoded ion channel is also thoughtto play a role in mediating the HIV life cycle. The present invention isnot limited to treating HIV infection via the mechanism of inhibitingthe HIV life cycle and, in particular, HIV replication. Rather, thepresent invention should be understood to encompass any mechanism bywhich the compounds of the present invention exert their anti-viralactivity and may include inhibition of HIV viability or functionalactivity. This also applies to HCV, Coronaviruses, and to other viruses.

Reference to the “functional activity” of a virus should be understoodas a reference to any one or more of the functions which a virusperforms or is involved in.

Reference to the “viral replication” should be understood to include anyone or more stages or aspects of the viral life cycle, such asinhibiting the assembly or release of virions. Ion channel mediation ofviral replication may be by direct or indirect means. Said ion channelmediation is by direct means if the ion channel interacts directly withthe virion at any one or more of its life cycle stages. Said ion channelmediation is indirect if it interacts with a molecule other than thoseof the virion, which other molecule either directly or indirectlymodulates any one or more aspects or stages of the viral life cycle.Accordingly, the method of the present invention encompasses themediation of viral replication via the induction of a cascade of stepswhich lead to the mediation of any one or more aspects or stages of theviral life cycle.

Reference to “down-regulating” ion channel functional activity, shouldbe understood as a reference to the partial or complete inhibition ofany one or more aspects of said activity by both direct and indirectmechanisms. For example, a suitable agent may interact directly with anion channel to prevent replication of a virus or, alternatively, may actindirectly to prevent said replication by, for example, interacting witha molecule other than an ion channel. A further alternative is that saidother molecule interacts with and inhibits the activity of the ionchannel.

Screening for molecules that have antiviral activity can be achieved bythe range of methodologies described herein.

Reference to a “cell” infected with a virus should be understood as areference to any cell, prokaryotic or eukaryotic, which has beeninfected with a virus. This includes, for example, immortal or primarycell lines, bacterial cultures and cells in situ. In a suitablescreening system for antiviral compounds, the preferred infected cellswould be macrophages/monocytes or hepatocytes/lymphoid cells infectedwith either HIV or HCV respectively.

Without limiting the present invention to any one theory or mode ofaction, the compounds of the present invention are thought to inhibitviral replication or virion release from cells by causing ion channels,namely VPU of HIV, the E protein of SARS and other Coronaviruses, or P7of HCV to become blocked. The present invention encompasses antiviralcompounds that are substituted acylguanidines.

The present invention also includes the use of compounds5-(N,N-hexamethylene)amiloride and 5-(N,N-dimethyl)-amiloride in thecontrol of viral replication and/or growth other than HIV.

The subject of the viral inhibition is generally a mammal such as butnot limited to human, primate, livestock animal (e.g. sheep, cow, horse,donkey, pig), companion animal (e.g. dog, cat), laboratory test animal(e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animal(e.g. fox, deer). Preferably, the subject is a human or primate. Mostpreferably, the subject is a human.

The method of the present invention is useful in the treatment andprophylaxis of viral infection such as, for example, but not limited toHIV infection, HCV infection and other viral infections. For example,the antiviral activity may be effected in subjects known to be infectedwith HIV in order to prevent replication of HIV thereby preventing theonset of AIDS. Alternatively, the method of the present invention may beused to reduce serum viral load or to alleviate viral infectionsymptoms. Similarly, antiviral treatment may be effected in subjectsknown to be infected with, for example, HCV, in order to preventreplication of HCV, thereby preventing the further hepatocyteinvolvement and the ultimate degeneration of liver tissue.

The method of the present invention may be particularly useful either inthe early stages of viral infection to prevent the establishment of aviral reservoir in affected cells or as a prophylactic treatment to beapplied immediately prior to or for a period after exposure to apossible source of virus.

Reference herein to “therapeutic” and “prophylactic” is to be consideredin their broadest contexts. The term “therapeutic” does not necessarilyimply that a mammal is treated until total recovery. Similarly,“prophylactic” does not necessarily mean that the subject will noteventually contract a disease condition. Accordingly, therapy andprophylaxis include amelioration of the symptoms of a particularcondition or preventing or otherwise reducing the risk of developing aparticular condition. The term “prophylaxis” may be considered asreducing the severity of onset of a particular condition. Therapy mayalso reduce the severity of an existing condition or the frequency ofacute attacks.

In accordance with the methods of the present invention, more than onecompound or composition may be co-administered with one or more othercompounds, such as known anti-viral compounds or molecules. By“co-administered” is meant simultaneous administration in the sameformulation or in two different formulations via the same or differentroutes or sequential administration by the same or different routes. By“sequential” administration is meant a time difference of from seconds,minutes, hours or days between the administration of the two or moreseparate compounds. The subject antiviral compounds may be administeredin any order.

Routes of administration include but are not limited to intravenously,intraperitionealy, subcutaneously, intracranialy, intradermally,intramuscularly, intraocularly, intrathecaly, intracerebrally,intranasally, transmucosally, by infusion, orally, rectally, via ivdrip, patch and implant. Intravenous routes are particularly preferred.

Compositions suitable for injectable use include sterile aqueoussolutions (where water soluble) and sterile powders for theextemporaneous preparation of sterile injectable solutions. The carriercan be a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol and liquidpolyethylene glycol, and the like), suitable mixtures thereof andvegetable oils. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed by, forexample, filter sterilization or sterilization by other appropriatemeans. Dispersions are also contemplated and these may be prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, a preferredmethod of preparation includes vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution.

When the active ingredients are suitably protected, they may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or it may be enclosed in hard or soft shell gelatincapsule, or it may be compressed into tablets. For oral therapeuticadministration, the active compound may be incorporated with excipientsand used in the form of ingestible tablets, buccal tablets, troches,capsules, elixirs, suspensions, syrups, wafers, and the like. Suchcompositions and preparations should contain at least 0.01% by weight,more preferably 0.1% by weight, even more preferably 1% by weight ofactive compound. The percentage of the compositions and preparationsmay, of course, be varied and may conveniently be between about 1 toabout 99%, more preferably about 2 to about 90%, even more preferablyabout 5 to about 80% of the weight of the unit. The amount of activecompound in such therapeutically useful compositions in such that asuitable dosage will be obtained. Preferred compositions or preparationsaccording to the present invention are prepared so that an oral dosageunit form contains between about 0.1 ng and 2000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain thecomponents as listed hereafter: A binder such as gum, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, lactose or saccharin may be added or a flavouringagent such as peppermint, oil of wintergreen, or cherry flavouring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both. A syrup or elixir may contain the activecompound, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavouring such as cherry or orange flavour.Any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound(s) may be incorporated intosustained-release preparations and formulations.

The present invention also extends to forms suitable for topicalapplication such as creams, lotions and gels. In such forms, theanti-clotting peptides may need to be modified to permit penetration ofthe surface barrier. Procedures for the preparation of dosage unit formsand topical preparations are readily available to those skilled in theart from texts such as Pharmaceutical Handbook. A Martindale CompanionVolume Ed. Ainley Wade Nineteenth Edition The Pharmaceutical PressLondon, CRC Handbook of Chemistry and Physics Ed. Robert C. Weast Ph D.CRC Press Inc.; Goodman and Gilman's; The Pharmacological basis ofTherapeutics. Ninth Ed. McGraw Hill; Remington; and The Science andPractice of Pharmacy. Nineteenth Ed. Ed. Alfonso R. Gennaro MackPublishing Co. Easton Pa.

Pharmaceutically acceptable carriers and/or diluents include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, use thereof in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the novel dosageunit forms of the invention are dictated by and directly dependent on(a) the unique characteristics of the active material and the particulartherapeutic effect to be achieved and (b) the limitations inherent inthe art of compounding.

Effective amounts contemplated by the present invention will varydepending on the severity of the pain and the health and age of therecipient. In general terms, effective amounts may vary from 0.01 ng/kgbody weight to about 100 mg/kg body weight.

Alternative amounts include for about 0.1 ng/kg body weight about 100mg/kg body weight or from 1.0 ng/kg body weight to about 80 mg/kg bodyweight.

The subject of the viral inhibition is generally a mammal such as butnot limited to human, primate, livestock animal (e.g. sheep, cow, horse,donkey, pig), companion animal (e.g. dog, cat), laboratory test animal(e.g. mouse, rabbit, rat, guinea pig, hamster), captive wild animal(e.g. fox, deer). Preferably, the subject is a human or primate. Mostpreferably, the subject is a human.

The methods of the present invention is useful in the treatment andprophylaxis of viral infection such as, for example, but not limited toHIV infection, HCV infection and other viral infections. For example,the antiviral activity may be effected in subjects known to be infectedwith HIV in order to prevent replication of HIV thereby preventing theonset of AIDS. Alternatively, the methods of the present invention maybe used to reduce serum viral load or to alleviate viral infectionsymptoms. Similarly, antiviral treatment may be effected in subjectsknown to be infected with, for example, HCV, in order to preventreplication of HCV, thereby preventing the further hepatocyteinvolvement and the ultimate degeneration of liver tissue.

The methods of the present invention may be particularly useful eitherin the early stages of viral infection to prevent the establishment of aviral reservoir in affected cells or as a prophylactic treatment to beapplied immediately prior to or for a period after exposure to apossible source of virus.

The present invention will now be described in more detail withreference to specific but non-limiting examples describing studies ofviral membrane ion channels and screening for antiviral activity. Someexamples involve the use of the SARS virus. It will be clear from thedescription herein that other lentiviruses, and coronaviruses and othercompounds may be used effectively in the context of the presentinvention. It is to be understood, however, that the detaileddescription is included solely for the purpose of exemplifying thepresent invention. It should not be understood in any way as arestriction on the broad description of the invention as set out above.

EXAMPLE 1 Synthesis of the Compounds of the Invention

The compounds of the present invention may be made from thecorresponding acid chlorides or methyl esters as shown in Scheme 1. Bothof these methods are well described in the literature.

The following examples show synthetic schemes for some compounds of theinvention.

EXAMPLE 2 Synthesis of Cinnamoylguanidine from Cinnamic Acid CinnamoylChloride

To a solution of trans-cinnamic acid (1.50 g, 10.12 mmol) in dry benzene(30 mL) containing a drop of N,N-dimethylformamide was added oxalylchloride (5.14 g, 40.5 mmol) causing the solution to effervesce. Afterrefluxing for 2 h, the solution was evaporated to dryness under reducedpressure. The resulting solid was dissolved in dry tetrahydrofuran (20mL) and added slowly to a solution of guanidine hydrochloride in 2Maqueous sodium hydroxide (25 mL). The reaction was stirred at roomtemperature for 1 h then extracted with ethyl acetate (3×50 mL). Thecombined extracts were dried over magnesium sulfate and evaporated togive an orange oil. The crude product was purified by columnchromatography. Elution with 10% to 20% methanol in dichloromethane gaveCinnamoylguanidine as a cream solid (0.829 g, 43%).

EXAMPLE 3 Synthesis ofN-amidino-3-amino-5-phenyl-6-chloro-2-pyrazinecarboxamide

Part 1

To a solution of methyl 3-amino-5,6-dichloro-2-pyrazinecarboxylate(0.444 g, 2.0 mmol) in tetrahydrofuran (5 mL)/water (10 mL)/toluene (20mL) was added phenyl boronic acid (0.536 g, 4.4 mmol), sodium carbonate(0.699 g, 6.6 mmol) and tetrakis(triphenylphosphine)-palladium(0) (0.116g, 0.10 mmol). The reaction was evacuated and purged with nitrogenseveral times before being refluxed for 6 h. The organic layer wasseparated and the aqueous layer extracted with toluene (3×20 mL).

The combined organic extracts were dried over magnesium sulfate,filtered and evaporated under reduced pressure to give methyl3-amino-6-chloro-5-phenyl-2-pyrazinecarboxylate as a yellow solid (0.43g, 82%).

Part 2

To a solution of sodium (0.040 g, 1.74 mmol) dissolved in methanol (5mL) was added guanidine hydrochloride (0.258 g, 2.70 mmol) and themixture refluxed for 30 min after which it was filtered. To the filtratewas added methyl 3-amino-6-chloro-5-phenyl-2-pyrazinecarboxylate (0.264g, 1.0 mmol) in N,N-dimethylformamide (5 mL) and the solution heated at75.0 for 12 h. The solvent was removed under reduced pressure and theresidue chromatographed on silica gel eluting with 1% triethylamine/5%methanol/dichloromethane. The resulting solid was suspended inchloroform, filtered and dried under high vacuum to giveN-Amidino-3-amino-5-phenyl-6-chloro-2-pyrazinecarboxamide as a yellowsolid (0.04 g, 14%).

EXAMPLE 4 Synthesis of hexamethyleneimino-6-phenyl-2-pyrazinecarboxamide

Part 1

To a solution of methyl 3-amino-5,6-dichloro-2-pyrazinecarboxylate (1.11g, 5.0 mmol) in tetrahydrofuran (50 mL) was added hexamethyleneimine(1.49 g, 15.0 mmol) and the reaction was refluxed for 1 h. The reactionwas allowed to cool and the solid hexamethyleneimine hydrochlorideremoved by filtration. The filtrate was evaporated and the residuechromatographed over silica gel. Elution with dichloromethane gavemethyl 3-amino-6-chloro-5-hexamethyleneimino-2-pyrazinecarboxylate as anoff-white solid (1.20 g, 85%).

Part 2

To a solution of methyl3-amino-6-chloro-5-hexamethyleneimino-2-pyrazinecarboxylate (0.350 g,1.23 mmol) in dimethylsulfoxide (5 mL) was added phenyl boronic acid(0.166 g, 1.35 mmol), potassium carbonate (0.511 g, 3.70 mmol) and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)-dichloromethanecomplex (0.041 g, 0.05 mmol). The reaction was heated at 90° C. for 16 hbefore being poured into water (50 mL) and extracted with ethyl acetate(3×50 mL). The combined extracts were dried over magnesium sulfate,filtered and evaporated to give a brown oil which was purified bychromatography on silica gel. Elution with dichloromethane followed by10% ethyl acetate/dichloromethane gave methyl3-amino-5-hexamethyleneimino-6-phenyl-2-pyrazinecarboxylate as a yellowsolid (0.309 g, 77%).

Part 3.

To a solution of sodium (0.090 g, 6.17 mmol) dissolved in methanol (8mL) was added guanidine hydrochloride (0.598 g, 6.26 mmol) and themixture was refluxed for 30 min after which it was filtered. To thefiltrate was added methyl3-amino-5-hexamethyleneimino-6-phenyl-2-pyrazinecarboxylate (0.310 g,0.95 mmol) in tetrahydrofuran (10 mL) and the solution refluxed for 72h. The solvent was removed under reduced pressure and the residuechromatographed on silica gel. Elution with 5% methanol/dichloromethanegaveN-amidino-3-amino-5-hexamethyleneimino-6-phenyl-2-pyrazinecarboxamide asa yellow solid (0.116 g, 35%).

EXAMPLE 5 Viral Studies

Construction of Recombinant Plasmids Containing Open Reading FramesEncoding Various Virus Proteins.

Complimentary DNA (cDNA) fragments for the various viral proteins listedin Table 2 were obtained either by PCR amplification from a parentalvirus genome clone, or by direct chemical synthesis of thepolynucleotide sequence. For example, the open reading frame encodingVpu (FIG. 1a ) was amplified by PCR from a cDNA clone of an Nde Ifragment of the HIV-1 genome (isolate HXB2, McFarlane Burnet Centre,Melbourne, Australia) as follows: Native Pfu DNA polymerase (Stratagene;0.035 U//Il) was chosen to catalyse the PCR reaction to minimisepossible PCR introduced errors by virtue of the enzyme's proofreadingactivity. The 5′, sense, primer

-   -   AGTAGGATCCATGCAACCTATACC (<400>2) (SEQ ID NO: 2) introduces a        BamH1 site        (underlined) for cloning in-frame with the 3′ end of the GST        gene in p2GEX (41). This primer also repairs the start codon        (bold T replaces a Q of the vpu gene which is a threonine codon        in the HXB2 isolate. The 3′, antisense, primer

-   TCTGGAATTCTACAGATCATCAAC (<400>3) (SEQ ID NO: 3) introduces an EcoR1    site    (underlined) to the other end of the PCR product to facilitate    cloning. After 30 cycles of 94° C. for 45 sec, 55° C. for 1 min and    72° C. for 1 min in 0.5 ml thin-walled eppendorf tubes in a    Perkin-Elmer thermocycler, the 268 bp fragment was purified,    digested with BamH1 and EcoR1 and ligated to p2GEX prepared by    digestion with the same two enzymes. The resultant recombinant    plasmid is illustrated in FIG. 1b . The entireVpu open reading frame    and the BamH1 and EcoR1 ligation sites were sequenced by cycle    sequencing, using the Applied Biosystems dye-terminator kit, to    confirm the DNA sequence. Other cDNAs were synthesised for us using    state of the art methods by GenScript Corporation (New Jersey, USA).    Codon sequences were optimised for expression in bacterial, insect    or mammalian cells, as appropriate. Restriction endonuclease enzyme    recognition sites were incorporated at the 5′ and 3′ ends of the    synthetic cDNAs to facilitate cloning into plasmid expression    vectors, pcDNA3.1, pFastBac and pPL451 for expression of the encoded    virus proteins in mammalian, insect or bacterial cells,    respectively.

Standard techniques of molecular biology were used in cloningexperiments. For example, to prepare the Vpu open reading frame forinsertion into the pPL451 expression plasmid, p2GEXVpu was firstdigested with BamHI and the 5′ base overhang was filled in the KlenowDNA polymerase in the presence of dNTPs. The Vpu-encoding fragment wasthen liberated by digestion with EcoR1, purified from an agarose gel andligated into pPL451 which had been digested with HpaI and EcoR1. Westernblots subsequently confirmed that the pPLVpu construct (FIG. 1c )expressed Vpu after induction of cultures at 42° C. to inactivate thec1857 repressor of the PR and PL promoters.

TABLE 2 Source of viral cDNA or peptide sequences. Strain or SequenceTarget Protein Source organism Accession number Vpu HIV-1 strain HXB2SARS-CoV E protein SARS coronavirus P59637 HCV p7 Hepatitus C virus H771a NP_751922 MHV-E protein Murine hepatitis virus NP_068673 229E Eprotein Human coronavirus 229E NP_073554 Dengue M protein Dengue virustype 1 Strain Singapore S275/90

EXAMPLE 6 Purification of Recombinant Vpu from E. Coli

Cultures of E. coli strain XLI-blue cells containing p2GEXVpu were grownat 30° C. with vigorous aeration in LB medium supplemented with glucose(6 g/L) and ampicillin (50 mg/L) to a density of approximately 250 Klettunits, at which time IPTG was added to a final concentration of 0.01 mMand growth was continued for a further 4 hr. The final culture densitywas approximately 280 Klett units. Since early experiments revealed thatthe majority of expressed GST-Vpu fusion protein was associated withboth the cell debris and 30 membrane fractions, the method ofVaradhachary and Maloney (Varadhachary and Maloney, 1990) was adopted toisolate osmotically disrupted cell ghosts (combining both cell debrisand membrane fractions) for the initial purification steps. Cells wereharvested, washed, weighed and resuspended to 10 ml/g wet weight inMTPBS containing DTT (ImM) and MgC1₂ (10 mM). Lysozyme (0.3 mg/ml;chicken egg white; Sigma) was added and incubated on ice for 30 min withgentle agitation followed by 5 min at 37° C. The osmotically sensitisedcells were pelleted at 12,000 g and resuspended to the original volumein water to burst the cells. The suspension was then made up to1×MTPBS/DTT using a 10× buffer stock and the ghosts were isolated bycentrifugation and resuspended in MTPBS/DTT to which was thensequentially added glycerol (to 20% wt/vol) and CHAPS (to 2% wt/vol) togive a final volume of one quarter the original volume. This mixture wasstirred on ice for 1 hr and then centrifuged at 400,000 g for 1 hr toremove insoluble material. The GST-Vpu fusion protein was purified fromthe detergent extract by affinity chromatography on a glutathioneagarose resin (Sigma). The resin was thoroughly washed in 50 mM Tris pH7.5 containing glycerol (5%), DTT (1 mM), and CHAPS (0.5%) (Buffer A)and then the Vpu portion of the fusion protein was liberated and elutedfrom the resin-bound GST by treatment of a 50% (v/v) suspension of thebeads with human thrombin (100 U/ml; 37° C. for 1 hr). PMSF (0.5 mM) wasadded to the eluant to eliminate any remaining thrombin activity. ThisVpu fraction was further purified on a column of MA7Q anion exchangeresin attached to a BioRad HPLC and eluted with a linear NaCl gradient(0-2M) in buffer A.

The Vpu was purified to homogeneity—as determined on silver stainedgels—on an immunoaffinity column as follows: HPLC fractions containingVpu were desalted on a NAP 25 column (Pharmacia) into buffer A and thenmixed with the antibody-agarose beads for 1 hr at room temperature. Thebeads were washed thoroughly and Vpu was eluted by increasing the saltconcentration to 2M. Protein was quantitated using the BioRad dyebinding assay.

EXAMPLE 7 Expression and Purification of Vpu in E. Coli

The plasmid p2GEXVpu (FIG. 1) was constructed to create an in-frame genefusion between the GST and Vpu open-reading frames. This system enabledIPTG-inducible expression of the Vpu polypeptide fused to the C-terminusof GST and allowed purification of the fusion protein by affinitychromatography on glutathione agarose.

Optimal levels of GST-Vpu expression were obtained by growing thecultures at 30° C. to a cell density of approximately 250-300 Klettunits and inducing with low levels of IPTG (0.01 mM). To purify theGST-Vpu, a combined cellular fraction containing the cell debris andplasma membrane was prepared by lysozyme treatment of the induced cellsfollowed by a low-speed centrifugation. Approximately 50% of the GST-Vpuprotein could be solubilised from this fraction using the zwitterionicdetergent CHAPS. Affinity chromatography using glutathione-agarose beadswas used to enrich the fusion protein and thrombin was used to cleavethe fusion protein at the high affinity thrombin site between the fusionpartners, liberating Vpu (FIG. 2A). In fractions eluted from the anionexchange column Vpu was the major protein visible on silver stained gels(FIG. 2B, lane 1). Finally, Vpu was purified to apparent homogeneity onan immunoaffinity column (FIG. 2B, lane 2). The N-terminal amino acidsequence of the protein band (excised from SDS-PAGE gels) correspondingto the immunodetected protein confirmed its identity as Vpu.

EXAMPLE 8 Reconstitution of Vpu in Phospholipid Vesicles

Proteoliposomes containing Vpu were prepared by the detergent dilutionmethod (New, 1990). A mixture of lipids (PE:PC:PS; 5:3:2; 1 mg totallipid) dissolved in chloroform was dried under a stream of nitrogen gasand resuspended in 0.1 ml of potassium phosphate buffer (50 mM pH 7.4)containing DTT (ImM). A 25 μl aliquot containing purified Vpu was added,followed by octylglucoside to a final concentration of 1.25% (wt/vol).This mixture was subject to three rounds of freezing in liquid nitrogen,thawing and sonication in a bath type sonicator (20-30 sec) and was thenrapidly diluted into 200 volumes of the potassium phosphate buffer.Proteoliposomes were collected by centrifugation at 400,000 g for 1 hrand resuspended in approximately 150 μl of phosphate buffer.

EXAMPLE 9 Assaying Vpu Ion Channel Activity

Purified Vpu was tested for its ability to induce channel activity inplanar lipid bilayers using standard techniques as described elsewhere(Miller, 1986; and Piller et al, 1996). The solutions in the CIS andTRANS chambers were separated by a Delrin™ plastic wall containing asmall circular hole of approximately 100 μm diameter across which alipid bilayer was painted so as to form a high resistance electricalseal. Bilayers were painted from a mixture (8:2) ofpalmitoyl-oleoly-phosphatidyl-ethanolamine andpalmitoyl-oleolyphosphatidyl-choline (Avanti Polar Lipids, Alabaster,Ala.) in n-decane. The solutions in the two chambers contained MESbuffer (10 mM, pH 6.0) to which various NaC1 or KC1 concentrations wereadded. Currents were recorded with an Axopatch™ 200 amplifier. Theelectrical potential between the two chambers could be manipulatedbetween +/−200 mV (TRANS relative to grounded CIS). Aliquots containingVpu were added to the CIS chamber either as a detergent solution orafter incorporation of the protein into phospholipid vesicles. Thechamber was stirred until currents were observed.

EXAMPLE 10 Vpu Forms Ion Channels in Lipid Bilayers

To assay for ion-channel formation by Vpu, reconstitution into planarlipid bilayers was performed. When samples (containing between 7 and 70ng of protein) of purified recombinant Vpu were added to the 1 ml ofbuffer in the CIS chamber of the bilayer apparatus, current fluctuationswere detected after periods of stirring that varied from 2 to 30 min(FIG. 3). This time taken to observe channel activity approximatelycorrelated with the amount of protein added to the chamber. No channelswere detected when control buffer aliquots or control lipid vesicleswere added to the CIS chamber. In those control experiments the chamberscould be stirred for more than an hour without appearance of channelactivity.

EXAMPLE 11 Properties of the Vpu Channels

Channel activity was observed in over 40 individual experiments with Vpusamples prepared from five independent purifications. In differentexperiments, the amplitude of the currents varied over a large rangeand, again, seemed to approximately correlate with the amount of proteinadded. The smallest and largest channels measured had conductances of 14pS and 280 pS, respectively. The channels were consistently smaller whenlipid vesicles containing Vpu were prepared and fused to the bilayerrather than when purified protein in detergent solution was added. Thismay be because the former method included treatment with highconcentrations of detergent and a dilution step that may have favouredthe breakdown of large aggregates into monomers.

The relationship between current amplitude and voltage was linear andthe reversal potential in solutions containing a ten-fold gradient ofNaCl (500 mM CIS; 50 mM TRANS) was +3 OmV (FIG. 3B). A similar reversalpotential was obtained when solutions contained KCI instead of NaCl. In5 experiments with either NaCl or KCI in the solutions on either side ofthe membrane, the average reversal potential was 31.0+/−1.2 mV (+/−SEM).This is more negative than expected for a channel selectively permeablefor the cations alone. Using ion activities in the Goldman-Hodgkin-Katzequation gives a P_(Na)/P_(cl) ratio of about 5.5 indicating that thechannels are also permeable to chloride ions. An attempt was made toreduce the anion current by substituting phosphate for chloride ions.When a Na-phosphate gradient (150 mM Na & 100 mM phosphate CIS; 15 mMNa⁺ & 10 mM phosphate TRANS, pH 6.8) was used instead of the NaClgradient, the reversal potential was 37.1+/−0.2 (+/−SEM, n=2) againindicating a cation/anion permeability ratio of about 5. (Forcalculations involving the phosphate solutions, the summed activities ofthe mono and bivalent anions were used and it was assumed that the twospecies were equally permeable). The current-voltage curve now exhibitedrectification that was not seen in the NaCl solutions. It can beconcluded that the channels formed by Vpu are equally permeably to Na⁺and K⁺ and are also permeable, though to a lesser extent, to chloride aswell as phosphate ions.

EXAMPLE 12 Bacterial Bio-Assay for Screening Potential IonChannel-Blocking Drugs

This bio-assay is based on the observation that expression of Vpu in E.coli results in an active Vpu channel located in the plasmalemma thatdissipates the transmembrane sodium gradient. As a consequence of thisVpu channel activity, metabolites whose accumulation within the cells ismediated by a sodium dependent co-transporter (for example proline oradenine) leak out of the cell faster than they can be synthesised sothat the metabolites' intracellular levels become limiting for growth ofthe cell. Thereby, an E. coli cell expressing Vpu is unable to grow inminimal drop-out media lacking adenine or proline. However, in thepresence of a drug that blocks the Vpu channel, the cell is once againable to re-establish its transmembrane sodium gradient—due to the actionof other ion pumps in the membrane—and the leakage of metabolites isprevented enabling growth. Experiments to demonstrate that Vpu can formsodium channels in the plasma membrane of E. coli were performed asfollows.

To express unfused Vpu in E. coli, the vpu open-reading frame was clonedinto the plasmid pPL451 to create the recombinant plasmid pPL-Vpu (FIG.1b ). In this vector the strong P_(L) and P_(R) lambda promoters areused to drive expression of Vpu under control of the temperaturesensitive c1857 represser, such that when grown at 30° C. expression istightly repressed and can be induced by raising the temperature tobetween 37° C. and 42° C. On agar plates, cells containing pPL-Vpu grewwhen incubated at 30° C. and 37° C. but not at 42° C., while controlstrains grew well at 42° C. Liquid cultures of cells containing pPL-Vpuwere grown at 30° C. to OD₆₀₀,=0.84 then moved to grow at 42° C. for twohours (the final cell density was OD₆₀₀,=0.75). The plasma membranefraction was prepared and western blotting, using an antibody thatspecifically binds to the C-terminus of Vpu, detected a single band atapproximately 16 kDa, indicating that Vpu was expressed and associatedwith the membranes (FIG. 2A, lane 5).

EXAMPLE 13 Cross-Feeding Experiments Reveal that Proline Leaks Out ofCells Expressing Vpu

Uptake of proline by E. coli is well characterised and active transportof the amino acid into the cells is known to use the sodium gradient asthe energy source (Yamato et al, 1994). To detect whether prolineleakage occurs, the following cross-feeing assay was used: A lawn of anE. coli strain auxotrophic for proline and methionine (Met⁻Pro⁻), wasseeded and poured as a soft agar overlay on minimal drop-out mediaplates lacking proline but containing methionine. Sterile porous filterdiscs were inoculated with a Met⁺Pro⁺ strain (XL-1 blue) containingeither the pPL451 control plasmid or pPL-Vpu and placed onto the softagar. The plates were then incubated at 37° C. or 30° C. for two days.After than time a halo growth of the Met⁻Pro⁻ strain was clearly visiblesurrounding the disc inoculated with the cells containing pPL-Vpuincubated at 37° C. (FIG. 4A). This growth can only be due to theleakage of proline from the Vpu-expressing cells on the disc. No suchleakage was apparent from the control strain at 37° C. nor around eitherstrain on plates grown at 30° C. (FIG. 4B).

In contrast to proline transport, the E. coli methionine permease isknown to belong to the ABC transporter family (Rosen, 1987) and hence beenergised by ATP. Identical crossfeeding experiments to those describedabove were set us except that the Met⁻Pro⁻ strain was spread on minimaldrop-out plates lacking methionine but containing proline. No growth ofthis strain was evident around any of the discs (FIG. 4C), indicatingthat methionine was not leaking out of the XL-1 blue cells even when Vpuwas being expressed.

EXAMPLE 14 E.Coli Cells Expressing Vpu Require Adenine in the ExternalMedium for Growth

It was observed that, due to an uncharacterised mutation in the adeninesynthesis pathway, growth of E. coli cells of the XL1-blue strainexpressing Vpu at 37° C. was dependant on the presence of adenine in themedium. This allowed the development of an even simpler bioassay for Vpuion-channel activity than the proline cross-feeding assay describedabove: A lawn of XL1-blue cells containing the pPL-Vpu plasmid is seededonto an agarose plate lacking adenine in the medium, small aliquots ofdrugs to be tested for inhibition of the Vpu channel are spotted ontothe agarose in discrete locations and the plates are incubated at 37° C.for a suitable period of time (12-36 hours). Halos of growth around aparticular drug application site indicate that the drug has inhibitedexpression of the Vpu ion channel activity that prevents growth in theabsence of the drug. (FIG. 5).

EXAMPLE 15 Assay of Compounds in Planar Lipid Bilayers for Vpu ChannelBlocking Activity

Compounds were characterized for their ability to block Vpu ion channelactivity reconstituted into planar lipid bilayers. Vpu N-terminalpeptide (residues 1-32) dissolved in trifluoroethanol was added to theCIS chamber of the bilayer apparatus and the solutions was stirred untilion currents were observed, indicating incorporation of one or more Vpuion channels into the bilayer. After recording the channel activity fora few minutes, drugs were added to the solutions in the CIS and TRANSchambers—with stirring—to a final concentration of 100 μM. Channelactivity was then recorded for at least a further three minutes and theeffect of drug addition on ion current was determined by comparing thechannel activity before and after drug addition. For each experiment,drug effect was classified into four categories: “Stong block”, ifcurrent was inhibited approximately 90-100%; “weak block”, approx.50-90% inhibition; “partial block”, <50%; and “no effect”. Experimentswere disregarded if currents larger than ±50 pA were generated afteraddition of Vpu N-peptide because in such cases it is possible thatnon-native peptide aggregates contribute to bilayer breakdown. Suchaggregates, by virtue of their disorganized structure may not bespecifically blocked by the drugs at the concentrations tested.

Table 3 summarises the results of the bilayer experiments. A noveloutcome of these experiments was the strong blocking of Vpu channelsobserved with Phenamil. Phenamil has a phenyl group derivative at theguanidine group of amiloride. Amiloride itself is not a blocker of Vpu,whereas addition of the hexamethylene group at the 5-position of thepyrazine ring created a structure (HMA) that blocks the channel atconcentrations as low as 25 μM. These new results with Phenamil,however, now show that a bulky hydrophobic derivative at the oppositeend of the molecule can also turn amiloride into an effective Vpuchannel blocker. Interestingly, benzamil, with a very similar structurewas much less effective at blocking the Vpu channel.

TABLE 3 Summary of Compounds Inhibiting the Vpu Ion Channel in BilayersNo. of Compound Expts. Results Phenamil 3 3x Strong block MIA 2 1xStrong block; 1x weak Benzamil 10 3x partial block; 7x no effect EIPA 33x weak block; HMA 1 1x Strong block;(5-Phenyl-penta-2,4-dienoyl)guanidine 6 6x strong block6-methoxy-2-naphthoylguanidine 5 5x strong block(2-Chlorocinnamoyl)guanidine 6 4x strong; 2x partial blocks3-(trifluoromethyl)cinnamoylguanidine 5 4x strong blocks; 1x no effectN-{5-[3-(5-Guanidino-pentyloxymethyl)- 4 3x strong block; 1x no effectbenzyloxy]-pentyl}-guanidine 4-phenylbenzoylguanidine 3 3x strong block3-methylcinnamoylguanidine 4 2x strong block; 2x partial(3-Chlorocinnamoyl)guanidine 4 2x strong block; 2x partialN-(3-phenylpropanoyl)-N′- 1 1x strong blocks phenylguanidine(3-Bromocinnamoyl)guanidine 3 3x partial-strong block5-tert-butylamino-amiloride 3 3x partial blockN-amidino-3-amino-5-phenyl-6-chloro-2- 3 3x partial blockpyrazinecarboxamide 3-methoxy-HMA 3 3x partial block5-(N-Methyl-N-isobutyl)amiloride 1 1x partial block5-(N-Ethyl-N-isopropyl)amiloride 1 1x partial block 2-napthoylguanidine7 7x weak block N,N′-bis(3phenylpropanoyl)-N″- 7 7x weak blockphenylguanidine cinnamoylguanidine 3 3x weak block(5-Phenyl-penta-2,4-dienoyl)guanidine 6 6x strong block

EXAMPLE 16 Compound Screening Using the Bacterial Bio-Assay for the VpuProtein

The halos of growth around the site of application of particulardrugs—as described in example 14—were given a score between zero and sixreflecting the size and density of the zone of bacterial cell growth.Scores greater than 3 represent strong inhibition of the Vpu protein;scores between 1.5 and 3 represent moderate inhibition and scoresbetween 0.01 and 1.5 represent fair inhibition.

Table 4 lists the scores for inhibition of Vpu protein in the bacterialbio-assay.

TABLE 4 Vpu Inhibition (score/# of times Compound tested)(3-Chlorocinnamoyl)guanidine 4.38/4  (3-Bromocinnamoyl)guanidine  4.3/24(2-Chlorocinnamoyl)guanidine 4.0/4 (2-Bromocinnamoyl)guanidine 3.7/23-(trifluoromethyl)cinnamoylguanidine 3.7/25-bromo-2-fluorocinnamoylguanidine 3.5/2 3-methylcinnamoylguanidine3.4/2 2-methylcinnamoylguanidine 3.1/2 2,3-dimethylcinnamoylguanidine3.1/2 cinnamoylguanidine 2.96/12 6-methoxy-2-naphthoylguanidine 2.9/4trans-3-(1-napthyl)acryloylguanidine 2.9/33,4-dichlorocinnamoylguanidine 2.9/3 2,6-dichlorocinnamoylguanidine2.88/2  4-phenylbenzoylguanidine 2.75/5  2-ethylcinnamoylguanidine2.75/2  (4-Chlorocinnamoyl)guanidine 2.7/5 2-napthoylguanidine  2.7/112,5-dimethylcinnamoylguanidine 2.69/2  3-isopropylcinnamoylguanidinehydrochloride 2.6/2 (5-Phenyl-penta-2,4-dienoyl)guanidine 2.56/2 3-phenylcinnamoylguanidine 2.54/3  (4-Bromocinnamoyl)guanidine 2.5/45-(3′-bromophenyl)penta-2,4-dienoylguanidine 2.5/23-(cyclohex-1-en-1-yl)cinnamoylguanidine 2.5/23-(trifluoromethoxy)cinnamoylguanidine 2.44/2 2-(trifluoromethyl)cinnamoylguanidine 2.4/2N,N′-bis(3phenylpropanoyl)-N″-phenylguanidine 2.25/3 2-ethoxycinnamoylguanidine 2.25/2 N-(3-phenylpropanoyl)-N′-phenylguanidine 2.21/3 4-(trifluoromethyl)cinnamoylguanidine 2.2/2(4-Methoxycinnamoyl)guanidine 2.13/3  2-t-butylcinnamoylguanidine2.13/2  4-methylcinnamoylguanidine 2.1/2 2-fluorocinnamoylguanidine2.1/2 2-phenylcinnamoylguanidine 2.1/2N-(6-Hydroxy-2-napthoyl)-N′-phenylguanidine 2.06/2 3-t-butylcinnamoylguanidine 2.06/2  3,4-difluorocinnamoylguanidine2.06/2  5-(N,N-hexamethylene)amiloride  1.9/313-fluorocinnamoylguanidine 1.9/2 5-bromo-2-methoxycinnamoylguanidine1.9/2 3-ethoxycinnamoylguanidine 1.9/23,4-(methylenedioxy)cinnamoylguanidine 1.88/2 (2-Methoxycinnamoyl)guanidine 1.7/4 2′4 DichloroBenzamil HCl 1.7/22,3,5,6,-tetramethylcinnamoylguanidine 1.6/23-(2-napthyl)acryloylguanidine 1.56/2  2-(1-napthyl)acetoylguanidine1.56/2  2,3-difluorocinnamoylguanidine 1.56/2 (3-Methoxycinnamoyl)guanidine 1.52/6  4-isopropylcinnamoylguanidine1.4/2 2,4,6-trimethylcinnamoylguanidine 1.4/2N-(cinnamoyl)-N′phenylguanidine 1.25/3 2-(cyclohex-1-en-1yl)cinnamoylguanidine 1.2/22-(2-napthyl)acetoylguanidine 1.19/2  (4-Hydroxycinnamoyl)guanidine1.1/2 4-phenylcinnamoylguanidine 1.1/2 4-fluorocinnamoylguanidine 1.1/2N,N′-bis-(cinnamoyl)-N″-phenylguanidine 0.94/2 (2-Furanacryloyl)guanidine 0.94/2  Phenamil methanesulfonate salt 0.9/5Benzamil hydrochloride 0.9/3 (3-Nitrocinnamoyl)guanidine 0.9/1Benzyoylguanidine 0.88/2  (4-Phenoxybenzoyl)guanidine 0.81/2 3-(trans-hept-1-en-1-yl)cinnamoylguanidine 0.81/2 5-(N-Methyl-N-isobutyl)amiloride 0.8/2 2-cyclohexylcinnamoylguanidine0.8/2 4-ethoxycinnamoylguanidine 0.69/2  2,4-dichlorocinnamolyguanidine0.63/2  5-(N-Ethyl-N-isopropyl)amiloride 0.6/3N-amidino-3-amino-5-hexamethyleneimino-6-phenyl- 0.6/22-pyrazinecarboxamide (a-Methylcinnamoyl)guanidine 0.6/2cinnamoylguanidine hydrochloride 0.6/2[(4-Chlorophenoxy-acetyl]guanidine 0.56/2 N-amidino-3-amino-5-phenyl-6-chloro-2-  0.5/11 pyrazinecarboxamide5-(4-fluorophenyl)amiloride 0.4/6(trans-2-Phenylcyclopropanecarbonyl)guanidine 0.4/2(2-Nitrocinnamoyl)guanidine 0.4/2 trans-3-Furanacryoylguanidine 0.38/2 1-napthoylguanidine 0.3/2 5-tert-butylamino-amiloride 0.2/73-methoxy-HMA 0.2/4 (3-phenylpropanoyl)guanidine 0.2/44-t-butylcinnamoylguanidine 0.19/2  5-(N,N-Dimethyl)amiloridehydrochloride 0.1/2 N,N′-Bis(3-phenylpropanoyl)guanidine 0.1/2N-Benzoyl-N′-cinnamoylguanidine 0.06/2  1-bromo-2-napthoylguanidine0.06/2 

EXAMPLE 17 Effect of Compounds on HIV Replication in Human Monocytes andMacrophages

Human monocytes were isolated from peripheral blood and cultured eitherfor 24 hr (one day old monocytes) or for 7 days to allow differentiationinto monocyte derived macrophages (MDM). These cells were then exposedto cell-free preparations of HIV isolates and allowed to absorb for 2 hrbefore complete aspiration of the medium, washing once with virus-freemedium and resuspension in fresh medium. The cells were exposed tovarious concentration of compound either 24 hr prior to infection orafter infection. Subsequent HIV replication, at various times afterinfection, was compared in cells exposed to drugs and in cells notexposed to drugs (controls). The progression and extent of viralreplication was assayed using either an HIV DNA PCR method (Fear et al,1998) or an ELISA method to quantitate p24 in culture supernatants(Kelly et al, 1998).

Table 5 provides examples of results obtained using this assay and testanti-viral compounds.

TABLE 5 Drug Percent of Conc. Positive Compound μM Control None -positive control 100% 4-phenylbenzoylguanidine 10 26 5 4 2.5 9 1. 2160.625 10 None - positive control 100% (3-Bromocinnamoyl)guanidine 10 3 51 2.5 9 1.25 59 0.625 116 None - positive control 100% 3-(trifluoro- 1011 methyl)cinnamoylguanidine 5 8 2.5 25 1.25 27 0.625 38 None - positivecontrol 100% 5-(N,N- 10 6 hexamethylene)amiloride 5 21 2.5 50 1.25 190.625 30

EXAMPLE 18 SARS Coronavirus

SARS E Protein Forms an Ion Channel

Peptide Synthesis

A peptide corresponding to the full-length SARS-CoV (isolate Tor2 andUrbani) E protein (MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLCAYCCNIVNVSLVKPTVYVYSRVKNLNSSEGVPDLLV) (SEQ ID NO: 4) and a second peptidecomprising the first 40 amino acids of the full length E protein whichcorrespond to the transmembrane domain (MYSFVSEETGTLIVNSVLLFLAFVVFLLVTLAILTALRLC) (SEQ ID NO: 5) were synthesized manually using FMOCchemistry and solid phase peptide synthesis The synthesis was done atthe Biomolecular Resource Facility (John Curtin School of MedicalResearch, ANU, Australia) using a Symphony^(R) Peptide Synthesiser fromProtein Technologies Inc.(Tucson, Ariz., USA) according to themanufacturers instructions.

EXAMPLE 19 Peptide Purification

Mass spectral analysis of the synthetic peptide revealed that thepreparation contained significant amounts of material with lower m/zratio than expected for the full-length product. The majority of theseare presumably truncated peptides generated during the peptide synthesisprocess. To enrich the full-length E protein, the following procedurewas used, which relies on differential solubility of the smallermolecules and full-length peptide. The crude preparation was suspendedat 12 mg/ml in 70% CH₃CN, 0.1% TFA and vortexed for 10 minutes. Thissuspension was centrifuged at 10,000 g for 10 minutes at 20° C. Thesupernatant was discarded and the insoluble fractions was extracted with70% CH3CN, 0.1% TFA, as above, two more times. The insoluble materialcontaining the E protein was dried using Speedvac an the weight of thefinal product was used to calculate the yield. The purified peptide wasanalysed by Bruker Omniflex MALDI-TOF mass spectrometry in HABA matrixat 2.5 mg/ml in methanol at a 1:1 ratio and spectra were obtained in thepositive linear mode. A clear peat at m/z ratio of 8,360.1 was seen asexpected for the calculated molecular weight of full-length E proteinand 4422.3 for the N-terminal E protein.

EXAMPLE 20 Planar Lipid Bilayers

The SARS virus E protein was resuspended at 1 mg/ml in2,2,2-trifluoroethanol. The SARS virus E protein's ability to form ionchannels was tested on a Warner (Warner instruments, Inc. 1125 DixwellAvenue, Hamden, Conn. 06514) bilayer rig as follows; A lipid mix of3:1:1, 1-Palmitoyl-2-oleolyl phosphatidyl Ethanolamine:1-Palmitoyl-2-oleolyl phosphatidyl Serine: 1-Palmitoyl-2-oleolylphosphatidyl choline in CHCl₃ was dried under N₂ gas and resuspended to50 mg/ml in n-decane. Bilayers were painted across a circular hole ofapproximately 100 μm diameter in a Delrin™ cup separating aqueoussolution in the CIS and TRANS chambers. The CIS chamber contained asolution of 500 mM NaCl or KCl, in a 5 mM HEPES buffer pH 7.2, the TRANSchamber contained a solution of 50 mM NaCl or KCl, in a 5 mM HEPESbuffer pH 7.2. Silver electrodes coated in chloride with 2% agarosebridges are placed in the CIS and TRANS chamber solutions. The SARS Eprotein full-length or N-terminal peptides (3-10 ug) were added to theCIS chamber, which was stirred until channel activity was detected. TheCIS chamber was earthed and the TRANS chamber was held at variousholding potentials ranging between +100 to −100 mV. Currents wererecorded using a Warner model BD-525D amplifier, filtered at 1 kHz,sampling at 5 kHz and digitally recorded on the hard disk of a PC usingsoftware developed in house.

Drugs to be tested for their ability to inhibit SARS E protein ionchannel activity were made up at 50 mM in a solution of 50% DMSO: 50%methanol. For experiments testing the ability of compounds to inhibit Eprotein ion channel activity, 100 μM to 400 μM of compound was added tothe CIS chamber while stirring for 30 seconds. Bilayer currents wererecorded before channel activity, during channel activity and after theaddition of the drug.

Among the compounds tested was cinnamoylguanidine (Bit036), a compoundwhich was shown in earlier experiments to be antiviral and to inhibition channel proteins from other viruses.

EXAMPLE 20.1 Polyacrylamide Gel Electrophoresis

Purified E protein was dissolved to 1 mg/ml, 5 mg/ml and 10 mg/ml in, 6M Urea, 10% Glycerol, 5% SDS, 500 mM DTT, 0.002% Bromophenol Blue, 62.5mM Tris HCl (pH 8.3). Peptides in solutions were heated at 100° C. for20 minutes before 30 μL samples were run on stacking gel 4-20%(Gradipore). SeeBlue® pre-stained standard (Invitrogen) was used formolecular weight markers.

EXAMPLE 20.2 Results

To test if the SARS E protein forms ion channels the purified syntheticpeptide was reconstituted into planar lipid bilayers (21). Typically, 3μg of SARS full-length E protein was added to the CIS chamber, whilestirring. This CIS chamber contained 500 mM NaCl and the TRANS chambercontained 50 mM NaCl. In 60 experiments, ion currents due to SARS Eprotein ion channel activity were observed after about 5-15 minutes ofstirring. Activity was detected more rapidly and reliably with a holdingpotential of approximately −100 mV across the bilayer. Currents recordedat −100 mV, (A) and at −60 mV (B) in one of these experiments are shownin FIG. 6. In that experiment the reversal potential was about +48 mVand the channel conductances were calculated to be 52 pS and 26 pS,respectively. This indicates that the current-voltage (IV) relationshipis not linear. In ten other experiments, where no protein was added tothe CIS chamber, no ion channel activity was detected, even afterrecording for over 1 hour.

FIG. 7a shows typical current traces recorded over a range of potentialsin NaCl solutions. In that experiment the direction of current flowreversed at +48 mV (FIG. 7b ). The IV curve shows that at the lowervoltages the average current flow across the bilayer is small but athigher potentials there is an increase in average current across thebilayer, resulting in a non-linear IV relationship. In seven independentexperiments, the average reversal potential was +48.3±2.3 mV (mean±1SEM), indicating that the channels were about 37 times more permeable toNa+ ion than to Cl⁻ ions. The reversal potential is close to the Na+equilibrium potential (+53 mV), therefore the channel is selective forNa+ ions. For these 7 experiments the channel conductance varied between95-164 pS; the average conductance was 130±13 pS.

SARS E protein ion channel is slightly less selectivity for K⁺ ions thanNa⁺ ions. FIG. 8b shows recording of currents in KCl solutions at arange of potentials. In this experiment the currents reversed at +31 mV.In seven similar experiments E protein ion channel average reversalpotential was +34.5±2.5 mV. Therefore the SARS E protein ion channel isabout 7.2 times more permeable to K⁺ ions than Cl⁻ ions. In sevenexperiments, the channel conductance varied ranging between 24-166 pS,the average conductance was 83.4±26 pS.

Similar results were obtained with a second synthetic peptide, whichcorresponded to the first forty N-terminal amino acids of the SARS Eprotein “N-terminal peptide” (21). The average reversal potential inNaCl solution in four experiments was +46.3±2.5 mV, indicating that theion channel formed by N-terminal peptide is about 25 times morepermeable to Na+ ion than to Cl− ions. The SARS E protein N-terminalpeptide was sufficient for the formation of ion channels with propertieslike those of the full length SARS E protein. Therefore, the selectivityfilter for the SARS E protein is most likely contained within the firstforty amino acids of the N-terminal.

SARS E protein N-terminal peptide also formed ion channels in KClsolution that were similarly selective for K+ ions compared to thefull-length E protein. In five independent experiments the averagechannel reversal potential was +39.5±3.6 mV, therefore the channel isabout 11 times more permeable to K⁺ ions than Cl⁻ ions.

SDS-PAGE of the purified full-length E protein peptide showed bandscorresponding to the full-length E protein (Data not shown). Largerbands of varying size up to about 20 kDa were detected, suggesting thatSARS E protein may form homo-oligomers.

EXAMPLE 21 SARS E Protein Ion Channel is Blocked by Cinnamoylguanidineand Other Compounds

E protein ion channel activity in NaCl solutions was significantlyreduced (p≥0.01, n=6 experiments) by addition of 100 to 200 μMcinnamoylguanidine to the CIS chamber. The average current across thebilayer was reduced to baseline by 100 μM cinnamoylguanidine. Inexperiments when E protein ion channels had higher conductance, 100 to200 μM cinnamoulguanidine reduced the average current across the bilayerabout 4 fold. Similarly, in four other experiments, 100 to 200 μMcinnamoylguanidine blocked channels formed by full-length E protein inKCl solutions. In two additional experiments, the SARS E proteinN-terminal peptide was blocked by 100 to 200 μM cinnamoylguanidine,demonstrating that the cinnamoylguanidine drug-binding site is locatedwithin the first forty amino acids of the E protein N-terminal domain.Other compounds tested in bilayers for their effect on the SARS Eprotein are shown in below in Table 6.

TABLE 6 % Reduction of average Compound current by 100 μM5-(N,N-hexamethylene)amiloride 91 ± 7 6-methoxy-2-naphthoylguanidine  92± 16 2′4 DichloroBenzamil HCl 78 ± 0N,N′-bis(3phenylpropanoyl)-N″-phenylguanidine 88 ± 6(3-Bromocinnamoyl)guanidine  87 ± 11 (2-Bromocinnamoyl)guanidine 88 ± 6trans-3-(1-napthyl)acryloylguanidine 66 ± 2

EXAMPLE 21.1 Results and Discussion

We have shown that SARS E protein can form ion channels in lipid bilayermembranes. The ion currents reversed at positive potentials, whichdemonstrates that E protein ion channels are selective for monovalentcations over monovalent anions. E protein Ion channels were about 37times more selective for Na+ ions over Cl−ions and about 7.2 times moreselective for K+ ions over Cl− ions. In over 60 experiments the Na+conductance of the E protein ion channel varied from as low as 26 pS toas high as 164 pS. SDS-PAGE showed that the E protein formshomo-oligomers, and we surmised that the larger conductances wereprobably due to aggregation of the E protein peptide leading to largerion channels or the synchronous opening of many ion channels. Singlechannel currents were observed in several experiments and from these thechannel conductance was calculated to be voltage dependent.

The first 40 amino acids of the N-terminal which contains thehydrophobic domain of the SARS virus E protein is sufficient for theformation of ion channels on planar lipid bilayers. The N-terminal Eprotein ion channel has the same selectivity and conductance as thefull-length E protein ion channel.

The SARS virus full length E protein ion channel activity and N-terminaldomain E protein ion channel activity on planar lipid bilayers in NaCland KCl solutions was inhibited by addition of between 100 μM to 200 μMcinnamoylguanidine to the CIS chamber. Inhibition or partial inhibitionof the E protein ion channel activity by cinnamoylguanidine has beenobserved in seven independent experiments in NaCl solution and fourindependent experiments in KCl solution.

All known coronaviruses encode an E protein with a hydrophobicN-terminus transmembrane domain therefore all coronaviruses E proteinscould form ion channels on planar lipid bilayers. This indicates thatthe E protein could be a suitable target for antiviral drugs andpotentially stop the spread of coronavirus from infected host cells.Drugs that block the E protein ion channel could be effective antiviraltherapy for the treatment of several significant human and veterinarycoronavirus diseases including SARS and the common cold.

EXAMPLE 22 Bacterial Bio-Assay for Screening Potential SARS-CoV EProtein Ion Channel-Blocking Drugs

SARS-CoV E Protein Ion Channel Inhibits Bacterial Cell Growth

A bio-assay of SARS-CoV E protein function in bacterial cells wasdeveloped. A synthetic cDNA fragment encoding SARS-CoV E protein wascloned into the expression plasmid pPL451, creating a vector in which Eprotein expression is temperature inducible, as described in Example 4.Inhibition of the growth of E. coli cells expressing E protein at 37° C.was observed as an indicator of p7 ion channel function dissipating thenormal Na+ gradient maintained by the bacterial cells.

EXAMPLE 23 Compound Screening Using the Bacterial Bio-Assay for SARSCoronavirus E Protein

The halos of growth around the site of application of particulardrugs—as described in example 14—were scored as described in example 15.

Table 7 lists the scores for inhibition of SARS-CoV E protein in thebacterial bio-assay.

TABLE 7 SARS E protein Inhibition (score/# of times Compound tested)2,3-difluorocinnamoylguanidine 4.50/1 3,4-dichlorocinnamoylguanidine4.15/2 4-t-butylcinnamoylguanidine 4.00/1 3-(2-napthyl)acryloylguanidine3.88/1 (3-Chlorocinnamoyl)guanidine 3.87/33-(cyclohex-1-en-1-yl)cinnamoylguanidine 3.75/12,5-dimethylcinnamoylguanidine 3.63/1trans-3-(1-napthyl)acryloylguanidine 3.38/24-isopropylcinnamoylguanidine 3.16/2 (3-Bromocinnamoyl)guanidine 3.15/27 6-methoxy-2-naphthoylguanidine 3.13/35-(N-Methyl-N-isobutyl)amiloride 3.13/2 3-phenylcinnamoylguanidine3.13/1 (2-Chlorocinnamoyl)guanidine  3.1/3 2′4 DichloroBenzamil HCl3.00/2 4-phenylcinnamoylguanidine 2.75/24-(trifluoromethyl)cinnamoylguanidine 2.75/13-(trifluoromethoxy)cinnamoylguanidine 2.71/13-(trifluoromethyl)cinnamoylguanidine 2.67/1 2-ethoxycinnamoylguanidine2.57/1 cinnamoylguanidine hydrochloride 2.50/14-ethoxycinnamoylguanidine 2.48/2 (2-Bromocinnamoyl)guanidine 2.47/32,6-dichlorocinnamoylguanidine 2.25/1 3,4,5-trimethoxycinnamoylguanidine2.25/1 5-tert-butylamino-amiloride 2.01/2 3-t-butylcinnamoylguanidine2.00/1 5-bromo-2-fluorocinnamoylguanidine 2.00/1(4-Chlorocinnamoyl)guanidine 1.94/2 2-t-butylcinnamoylguanidine 1.86/12-cyclohexylcinnamoylguanidine 1.83/1 6-Iodoamiloride 1.75/23-(trans-hept-1-en-1-yl)cinnamoylguanidine 1.71/1(4-Bromocinnamoyl)guanidine 1.69/2 (4-Hydroxycinnamoyl)guanidine 1.63/2N-(3-phenylpropanoyl)-N′-phenylguanidine 1.57/2(3-Nitrocinnamoyl)guanidine 1.51/2 3-fluorocinnamoylguanidine 1.50/12-(1-napthyl)acetoylguanidine 1.50/1 2-ethylcinnamoylguanidine 1.50/15-(N,N-Dimethyl)amiloride hydrochloride 1.38/2 2-napthoylguanidine1.38/2 5-(4-fluorophenyl)amiloride 1.38/12-(trifluoromethyl)cinnamoylguanidine 1.38/1N-(6-Hydroxy-2-napthoyl)-N′-phenylguanidine 1.35/3(trans-2-Phenylcyclopropanecarbonyl)guanidine 1.34/3N,N′-bis(3phenylpropanoyl)-N″-phenylguanidine 1.33/3 1-napthoylguanidine1.32/3 Benzamil hydrochloride 1.32/2 3-methoxy-HMA 1.25/14-methylcinnamoylguanidine 1.25/1 4-fluorocinnamoylguanidine 1.25/13,4-(methylenedioxy)cinnamoylguanidine 1.25/15-(N,N-hexamethylene)amiloride  1.2/3 N-(cinnamoyl)-N′phenylguanidine1.19/2 5-(N-Ethyl-N-isopropyl)amiloride 1.07/23-methylcinnamoylguanidine 1.00/1 2-methylcinnamoylguanidine 1.00/12,3,5,6,-tetramethylcinnamoylguanidine 1.00/1trans-3-Furanacryoylguanidine 0.88/2 (4-Methoxycinnamoyl)guanidine0.88/2 (2-Furanacryloyl)guanidine 0.82/2 (3-phenylpropanoyl)guanidine0.73/5 2-(2-napthyl)acetoylguanidine 0.71/1 cinnamoylguanidine 0.69/3(2-Methoxycinnamoyl)guanidine 0.69/2 [3-(3-Pyridyl)acryloyl]guanidine0.67/3 4-phenylbenzoylguanidine 0.63/2 2,4-dichlorocinnamolyguanidine0.63/2 (3-Methoxycinnamoyl)guanidine 0.63/2 2-fluorocinnamoylguanidine0.63/1 (4-Phenoxybenzoyl)guanidine 0.57/2 (a-Methylcinnamoyl)guanidine0.50/1 5-(3′-bromophenyl)penta-2,4-dienoylguanidine  0.5/1(5-Phenyl-penta-2,4-dienoyl)guanidine 0.44/2(Quinoline-2-carbonyl)guanidine 0.41/1 (Phenylacetyl)guanidine 0.32/3N,N′-Bis(amidino)napthalene-2,6-dicarboxamide 0.25/26-bromo-2-napthoylguanidine 0.25/1 1-bromo-2-napthoylguanidine 0.25/12-chloro-6-fluorocinnamoylguanidine 0.25/1[(4-Chlorophenoxy-acetyl]guanidine 0.19/2 Phenamil methanesulfonate salt0.13/2 N-Benzoyl-N′-cinnamoylguanidine 0.13/2N-(2-napthoyl)-N′-phenylguanidine 0.07/2

EXAMPLE 24 SARS Antiviral Assay for Testing Compounds AgainstReplication of SARS Coronavirus (SARS-CoV)

Compounds were tested against SARS-CoV (Hong Kong strain) using virusplaque purified three times in Vero cells. Stock virus was generated byinfecting Vero cells at MOI=1×TCID₅₀ per 100 cells.

EXAMPLE 24.1 Screening for Anti-Viral Activity Using the VirusMicrotitre Assay

Monolayers of Vero cells grown in 25 cm² flasks were infected at amultiplicity of 1:50 and treated immediately post infection withcompounds at two concentrations, 10 uM and 2 uM. A control infectedmonolayer remained untreated. Samples of culture media were taken at 48hours post infection. Two aliquots from each of the samples (titrations1 and 2) were serially log diluted and 12 replicates of log dilutions −4to −7 added to cells in microtitre plates. Four days later, wells in themicrotitre plates were scored for cytopathic effect (CPE) and thetitration values calculated based on the number of CPE positive wells atthe 4 dilutions. Control titres were 4.8 and 5.9 TCID₅₀×10⁶ (average5.35×10⁶)

EXAMPLE 25 Effect of Compounds in SARS CoV Antiviral Assay

Three selected compounds were tested for activity against SARS-CoVaccording to the method described in example 21. Fortrans-3-(1-napthyl)acryloylguanidine and cinnamoylguanidine a decreasein virus titre of approximately 80% was observed at a concentration of10 uM and a reduction of approximately 50% was seen to persist at 2 μMtrans-3-(1-napthyl)acryloylguanidine.

Table 8 provides Virus titration data presented as % of a control (SARSCoV grown for 48 hours in the absence of compounds).

TABLE 8 Compound Average Concentration TCID₅₀ Titre Name (uM) (×10⁶) (%control) cinnamoylguanidine 10 1.3 24 2 4.4 82 trans-3-(1- 10 1.15 22napthyl)acryloylguanidine 2 2.45 46 6-methoxy-2- 10 5.95 111naphthoylguanidine 2 6.35 118 Control 0 5.35 100

EXAMPLE 26 Human 229E Coronavirus

Synthesis and Purification of a Peptide Corresponding to the 229E-EProtein

A peptide corresponding to the full-length 229E-E protein (sequence:MFLKLVDDHALVVNVLLWCVVLIVILLVCITIIKLIKLCFTCHMFCNRTVYGPIKNVYHIYQSYMHIDPFPKRVIDF (SEQ ID NO: 6); accession number NP_073554) wassynthesized manually using FMOC chemistry and solid phase peptidesynthesis. The synthesis was done at the Biomolecular Resource Facility(John Curtin School of Medical Research, ANU, Australia) using aSymphonyR Peptide Synthesiser from Protein Technologies Inc. (Woburn,Mass., USA) according to the manufacturers instructions to giveC-terminal amides, the coupling was done with HBTU andhydroxybenzotriazole in N-methylpyrrolidone. Each of the synthesiscycles used double coupling and a 4-fold excess of the amino acids.Temporary α-N Fmoc-protecting groups were removed using 20% piperidinein DMF.

The crude synthetic peptide was purified using the ProteoPlus™ kit(Qbiogene inc. CA), following manufactures instructions. Briefly, thepeptides were diluted in loading buffer (60 mM Tris-HCl pH 8.3, 6M urea,5% SDS, 10% glycerol, 0.2% Bromophenol blue, ±100 mM β-mercaptoethanol)and run on 4-20% gradient polyacrylamide gels (Gradipore, NSW,Australia) in tris-glycine electrophoresis buffer (25 mM Tris, 250 mMglycine, 0.1% SDS). The peptides were stained with gel code blue(Promega, NSW) and the bands corresponding to the full-length peptidewere excised out of the gel.

The gel slice was transferred to the ProteoPLUS™ tube and filled withtris-glycine electrophoresis buffer. The tubes were emerged intris-glycine electrophoresis buffer and subjected to 100 volts forapproximately 1 hour. The polarity of the electric current was reversedfor 1 minute to increase the amount of protein recovered. The peptideswere harvested and centrifuged at 13,000 rpm for 1 minute. The purifiedpeptides were dried in a Speedvac and the weight of the final productwas used to calculate the yield.

EXAMPLE 27 229E-E Protein Forms Ion Channels in Planar Lipid Bilayers

Lipid bilayer studies were performed as described elsewhere (Sunstrom,1996; Miller, 1986). A lipid mixture ofpalmitoyl-oleoyl-phosphatidyl-ethanolamine,palmitoyl-oleoyl-phosphatidylserine andpalmitoyl-oleoyl-phosphatidylcholine (5:3:2) (Avanti Polar Lipids,Alabaster, Ala.) was used. The lipid mixture was painted onto anaperture of 150-200 μm in the wall of a 1 ml delrin cup. The apertureseparates two chambers, cis and trans, both containing salt solutions atdifferent concentrations. The cis chamber was connected to ground andthe trans chamber to the input of an Axopatch 200 amplifier. Normallythe cis chamber contained either 500 mM NaCl or 500 mM KCl and the trans50 mM NaCl or 50 mM KCl. The bilayer formation was monitoredelectrically by the amplitude of the current pulse generated by acurrent ramp. The potentials were measured in the trans chamber withrespect to the cis. The synthetic peptide was added to the cis chamberand stirred until channel activity was seen. The currents were filteredat 1000 Hz, digitized at 5000 Hz and stored on magnetic disk.

The 229E E synthetic peptide was dissolved in 2,2,2-trifluorethanol(TFE) at 0.05 mg/ml to 1 mg/ml. 10 μl of this was added to the cischamber (1 ml aqueous volume) of the bilayer apparatus, which wasstirred via a magnetic “flea”. Ionic currents, indicating channelactivity in the bilayer, were typically detected within 15-30 min. Afterchannels were detected the holding potential across the bilayer wasvaried between −100 mV and +100 mV to characterise the size and polarityof current flow and enable the reversal potential to be determined.

In 15 experiments where the cis chamber contained 500 mM NaCl solutionand the trans chamber contained 50 mM NaCl solution, the averagereversal potential of the channel activity was calculated to be 22±7(SEM) mV. In 13 experiments where the cis chamber contained 500 mM KClsolution and the trans chamber contained 50 mM KCl solution, the averagereversal potential of the channel activity was calculated to be 38±4(SEM) mV. These results indicate that the 229E E protein forms cationselective ion channels that are slightly more selective for K⁺ than forNa⁺ ions.

FIG. 9 shows examples of raw current data for the 229E E ion channel atvarious holding potentials (cis relative to trans) in asymmetrical KClsolutions (500/50 mM). The graph is a representative plot of averagebilayer current (pA; y-axis) versus holding potential (mV; x-axis).

EXAMPLE 28 Chemical Compounds Inhibit the Ion Channel Activity of the229E E Protein Synthetic Peptide

To test compounds for their ability to block or otherwise inhibit theion channel formed by 229E E protein, small aliquots of solutionscontaining the compounds were added to the aqueous solutions bathingplanar lipids in which the peptide channel activity had beenreconstituted and the effect of the compound addition on the ioniccurrents was recorded and measured.

Compound stock solutions were typically prepared at 500 mM in DMSO. Thissolution was further diluted to 50 mM, or lower concentration in 50%DMSO/50% methanol and 2 μl of the appropriately diluted compound wasadded to the cis and/or trans chambers to yield the desired finalconcentration.

In the example shown in FIG. 10, addition of 100 μM cinnamoylguanidineto the cis chamber greatly reduced current flow through the 229E E ionchannel.

EXAMPLE 29 Bacterial Bio-Assay for Screening Potential 229E-CoV EProtein Ion Channel-Blocking Drugs

229E-CoV E-Protein Ion Channel Inhibits Bacterial Cell Growth.

A bio-assay of 229E-CoV E-protein function in bacterial cells wasdeveloped. A synthetic cDNA fragment encoding 229E-CoV E-protein wascloned into the expression plasmid pPL451, creating a vector in which Eprotein expression is temperature inducible, as described in Example 4.Inhibition of the growth of E. coli cells expressing E protein at 37° C.was observed as an indicator of p7 ion channel function dissipating thenormal Na+ gradient maintained by the bacterial cells.

EXAMPLE 30 Compound Screening Using the Bacterial Bio-Assay for 229E-CoVE-Protein

The halos of growth around the site of application of particulardrugs—as described in example 14—were scored as described in example 15.

Table 9 list the scores for inhibition of 229E-CoV E-protein in thebacterial bio-assay.

TABLE 9 229E E protein Inhibition Compound (score)4-isopropylcinnamoylguanidine 4.9 3,4-dichlorocinnamoylguanidine 4.43-(trifluoromethoxy)cinnamoylguanidine 4.1 4-t-butylcinnamoylguanidine4.0 3-isopropylcinnamoylguanidine hydrochloride 4.03-t-butylcinnamoylguanidine 3.9 2-t-butylcinnamoylguanidine 3.9trans-3-(1-napthyl)acryloylguanidine 3.75-bromo-2-methoxycinnamoylguanidine 3.6 2,3-difluorocinnamoylguanidine3.3 3-(2-napthyl)acryloylguanidine 3.0 2-phenylcinnamoylguanidine 3.03-phenylcinnamoylguanidine 2.9 3-(cyclohex-1-en-1-yl)cinnamoylguanidine2.4 4-phenylbenzoylguanidine 2.3 3-(trifluoromethyl)cinnamoylguanidine2.3 (4-Phenoxybenzoyl)guanidine 2.34-(trifluoromethyl)cinnamoylguanidine 2.32-(cyclohex-1-en-1yl)cinnamoylguanidine 2.3 (4-Bromocinnamoyl)guanidine2.0 5-(N,N-hexamethylene)amiloride 1.9 1-napthoylguanidine 1.95-(4-fluorophenyl)amiloride 1.8 (5-Phenyl-penta-2,4-dienoyl)guanidine1.8 (3-Bromocinnamoyl)guanidine 1.7 2,5-dimethylcinnamoylguanidine 1.62-(trifluoromethyl)cinnamoylguanidine 1.5 6-methoxy-2-naphthoylguanidine1.4 (4-Chlorocinnamoyl)guanidine 1.4 (3-Methoxycinnamoyl)guanidine 1.45-bromo-2-fluorocinnamoylguanidine 1.4 5-(N,N-Dimethyl)amiloridehydrochloride 1.3 cinnamoylguanidine 1.3 (2-Methoxycinnamoyl)guanidine1.1 (a-Methylcinnamoyl)guanidine 1.0 4-phenylcinnamoylguanidine 1.02,6-dichlorocinnamoylguanidine 1.0 (2-Bromocinnamoyl)guanidine 0.92,4,6-trimethylcinnamoylguanidine 0.9(trans-2-Phenylcyclopropanecarbonyl)guanidine 0.8(3-Chlorocinnamoyl)guanidine 0.8 2-(1-napthyl)acetoylguanidine 0.82-ethylcinnamoylguanidine 0.8 2-cyclohexylcinnamoylguanidine 0.8(4-Hydroxycinnamoyl)guanidine 0.6 2-ethoxycinnamoylguanidine 0.63-methylcinnamoylguanidine 0.5 2-methylcinnamoylguanidine 0.53-fluorocinnamoylguanidine 0.5 cinnamoylguanidine hydrochloride 0.52,3-dimethylcinnamoylguanidine 0.5 2-fluorocinnamoylguanidine 0.44-fluorocinnamoylguanidine 0.4 3,4-difluorocinnamoylguanidine 0.45-tert-butylamino-amiloride 0.3 2-napthoylguanidine 0.3N,N′-Bis(amidino)napthalene-2,6-dicarboxamide 0.3N,N′-Bis(3-phenylpropanoyl)guanidine 0.3 4-methylcinnamoylguanidine 0.35-(3′-bromophenyl)penta-2,4-dienoylguanidine 0.32,3,5,6,-tetramethylcinnamoylguanidine 0.3 3-ethoxycinnamoylguanidine0.3 N,N′-bis(3phenylpropanoyl)-N″-phenylguanidine 0.1(4-Methoxycinnamoyl)guanidine 0.1 (2-Chlorocinnamoyl)guanidine 0.1(3-Nitrocinnamoyl)guanidine 0.1 4-ethoxycinnamoylguanidine 0.13,4,5-trimethoxycinnamoylguanidine 0.1 2-(2-napthyl)acetoylguanidine 0.1N-(3-phenylpropanoyl)-N′-phenylguanidine 0.1

EXAMPLE 31 Antiviral Assay for Testing Compounds Against Replication ofHuman Coronavirus 229E (229E)

To determine the antiviral activity of compounds against humancoronavirus 229E replication (ATCC VR-740), an assay measuring reductionin the number of plaques formed in monolayers of 229E infected MRC-5cells (human lung fibroblasts; ATCC CCL-171) was developed: First, avirus working stock was prepared by amplification in MRC-5 cells. Thiswas then used to infect confluent monolayers of MRC-5 cells grown in6-well tissue culture plates by exposure to the virus at an MOI ofapprox. 0.01 pfu/cell for 1 hour at 35° C. in 5% CO₂. The infectiveinoculum was removed and replaced with fresh medium (DMEM supplementedwith 10% fetal calf serum) containing various test concentrations ofcompounds or the appropriate level of solvent used for the compounds(control). Plates were subsequently incubated at 35° C. (in 5% CO₂) for3-5 days post infection, after which time culture supernatant wasremoved and the cells were stained with 0.1% crystal violet solution in20% ethanol for 10 minutes. Plaques were counted in all wells and thepercentage reduction in plaque number compared to solvent control wascalculated. Measurements were performed in duplicate to quadruplicatewells.

TABLE 10 Plaque Reduction (% control/# experiments) Compound 5 uM 2.5 uM1 uM 2-t-butylcinnamoylguanidine 100/1 100/3 050/34-isopropylcinnamoylguanidine 100/1 100/2 057/23,4-dichlorocinnamoylguanidine 100/3 099/4 086/3 3- 100/2 098/4 077/3(trifluoromethoxy)cinnamoylguanidine 2,6-dichlorocinnamoylguanidine100/1 097/3 066/2 2-(cyclohex-1-en- 100/1 097/3 021/11yl)cinnamoylguanidine 2-cyclohexylcinnamoylguanidine 070/1 097/2 089/25-bromo-2- 100/2 096/4 088/3 methoxycinnamoylguanidine2-phenylcinnamoylguanidine 100/1 096/3 100/15-(2′-bromophenyl)penta-2,4- 100/2 095/3 079/2 dienoylguanidine4-t-butylcinnamoylguanidine 100/1 095/3 084/3 3-phenylcinnamoylguanidine094/3 077/2 (3-Bromocinnamoyl)guanidine 100/2 093/3 072/2(4-Bromocinnamoyl)guanidine 094/1 091/3 073/25-(N,N-hexamethylene)amiloride 089/  091/2 033/1trans-3-(1-napthyl)acryloylguanidine 100/1 091/2 064/23-(2-napthyl)acryloylguanidine 100/1 091/2 062/22,4-dichlorocinnamolyguanidine 100/2 090/4 064/3(2-Nitrocinnamoyl)guanidine 085/2 090/2 046/2 3- 097/2 089/4 064/3(trifluoromethyl)cinnamoylguanidine 5-bromo-2-fluorocinnamoylguanidine100/1 088/3 063/2 4-methylcinnamoylguanidine 091/2 087/4 063/2(3-Chlorocinnamoyl)guanidine 100/1 086/3 009/1(4-Methoxycinnamoyl)guanidine 100/1 085/4 057/3(4-Chlorocinnamoyl)guanidine 100/2 084/2 051/23-fluorocinnamoylguanidine 095/1 083/3 051/2 3-(cyclohex-1-en-1- 100/1082/3 063/2 yl)cinnamoylguanidine (a-Methylcinnamoyl)guanidine 023/1082/1 036/2 2,3,5,6,- 098/2 079/4 064/3 tetramethylcinnamoylguanidine2-fluorocinnamoylguanidine 090/1 079/3 045/2 4- 100/1 079/1 052/1(trifluoromethyl)cinnamoylguanidine (3-Nitrocinnamoyl)guanidine 100/1079/1 045/1 2,5-dimethylcinnamoylguanidine 092/2 078/1 078/13-t-butylcinnamoylguanidine 100/1 077/4 030/3(3-Methoxycinnamoyl)guanidine 089/1 075/2 030/13-methylcinnamoylguanidine 095/1 074/3 044/13-isopropylcinnamoylguanidine 089/1 074/3 014/1 hydrochloride(2-Bromocinnamoyl)guanidine 095/2 072/2 043/2 3-ethoxycinnamoylguanidine100/1 072/3 057/1 (5-Phenyl-penta-2,4-dienoyl)guanidine 100/1 072/2069/1 (2-Chlorocinnamoyl)guanidine 095/2 072/2 040/24-ethoxycinnamoylguanidine 073/1 069/2 057/1 4-fluorocinnamoylguanidine100/1 067/3 034/2 3,4-difluorocinnamoylguanidine 085/1 065/3 042/2N-(3-phenylpropanoyl)-N′- 051/1 064/1 000/1 phenylguanidine2,4,6-trimethylcinnamoylguanidine 075/2 063/3 062/22-methylcinnamoylguanidine 074/2 063/3 053/3 (trans-2- 063/2 022/1Phenylcyclopropanecarbonyl)- guanidine [(E)-3-(4-Dimethylaminophenyl)-2-059/1 methylacryloyl]guanidine N-Benzoyl-N′-cinnamoylguanidine 056/14-phenylbenzoylguanidine 076/1 055/2 071/1 trans-3-Furanacryoylguanidine055/2 018/1 (4-Phenoxybenzoyl)guanidine 069/1 054/3 040/2(2-Methoxycinnamoyl)guanidine 051/1 053/2 024/1N-amidino-3-amino-5-phenyl-6- 074/2 052/2 038/1 chloro-2-pyrazinecarboxamide N-(cinnamoyl)-N′phenylguanidine 084/1 048/2 035/1cinnamoylguanidine 095/2 047/2 059/1 3,4- 084/1 046/1 019/1(methylenedioxy)cinnamoylguanidine N,N′-Bis(amidino)napthalene-2,6-045/1 dicarboxamide 2,3-dimethylcinnamoylguanidine 073/1 044/2 024/15-(3′-bromophenyl)penta-2,4- 044/1 dienoylguanidine N,N′-Bis(3- 041/1phenylpropanoyl)guanidine 3-methoxy-amiloride 029/2 039/3 022/22,3-difluorocinnamoylguanidine 036/1 1-napthoylguanidine 036/1(3-phenylpropanoyl)guanidine 036/1 6-methoxy-2-naphthoylguanidine  49/3030/4 5-(N,N-Dimethyl)amiloride 027/1 hydrochloride2-ethoxycinnamoylguanidine 027/1 2-napthoylguanidine 027/13,4,5-trimethoxycinnamoylguanidine 027/1 3-methoxy-HMA 027/1Benzyoylguanidine 026/1 2- 022/1 (trifluoromethyl)cinnamoylguanidineN-amidino-3,5-diamino-6-phynyl-2- 022/1 pyrazinecarboxamidecinnamoylguanidine hydrochloride 020/1 (Quinoline-2-carbonyl)guanidine015/3 019/3 006/2 (4-Hydroxycinnamoyl)guanidine 019/15-(4-fluorophenyl)amiloride 018/1 2-(1-napthyl)acetoylguanidine 018/1(2-Furanacryloyl)guanidine 018/1 [3-(3-Pyridyl)acryloyl]guanidine 018/1N-Cinnamoyl-N′,N′- 015/1 dimethylguanidineN-(2-napthoyl)-N′-phenylguanidine 011/1 2-(2-napthyl)acetoylguanidine009/1 N,N′-bis(3phenylpropanoyl)-N″- 009/1 phenylguanidine(Phenylacetyl)guanidine 009/1

EXAMPLE 32 Human OC43 Coronavirus

OC43 Antiviral Assay for Testing Compounds Against Replication of HumanCoronavirus OC43.

To determine the antiviral activity of compounds against humancoronavirus OC43 replication (ATCC VR-759), an ELISA assay was developedmeasuring the release of the viral N-protein into culture supernatantsfrom monolayers of OC43-infected MRC-5 cells (human lung fibroblasts;ATCC CCL-171): First, a virus working stock was prepared byamplification in MRC-5 cells. This was then used to infect confluentmonolayers of MRC-5 cells grown in 6-well tissue culture plates byexposure to the virus at an MOI of approx. 0.01 pfu/cell for 1 hour at35° C. in 5% CO₂. The infective inoculum was removed and replaced withfresh medium (DMEM supplemented with 10% fetal calf serum) containingvarious test concentrations of compounds or the appropriate level ofsolvent used for the compounds (control). Plates were subsequentlyincubated at 35° C. (in 5% CO₂) for 5 days post infection, after whichtime culture supernatant was harvested and cellular debris removed bycentrifugation at 5000×g for 10 minutes. For N-antigen detection, 100 μlsamples of clarified culture supernatant were added to duplicate wellsof a 96-well Maxi-Sorb plate; 100 μl of RIPA buffer was added per wellwith mixing and the plate was covered and incubated at 4° C. overnightto enable protein binding to the plastic wells. The next day, thecoating solution was discarded, wells were washed thoroughly with PBST,and blocking of unoccupied protein binding sites was performed byincubation in 1% BSA in PBS for 1.5 hours. The antibody recognising OC43N-protein was used at 1/800 dilution in PBS (1 hr at 37° C.) and thesecondary antibody (goat-anti-mouse alkaline phosphatase) was used forthe colour development reaction. Optical density of the wells was readat 405 nm and the effect of compounds determined by comparison of thelevel of signal in presence of compound to level of signal from thesolvent control.

EXAMPLE 33 Effect of Compounds in OC43 Antiviral Assay

Compounds were screened for activity against OC43 replication accordingto the method described in example 22. Results are shown in Table 11.

TABLE 11 Virus inhibition at Compound 2.5 uM 3-methylcinnamoylguanidine100 trans-3-(1-napthyl)acryloylguanidine 100 (3-Bromocinnamoyl)guanidine100 (2-Chlorocinnamoyl)guanidine 96 3,4-dichlorocinnamoylguanidine 903-(trifluoromethyl)cinnamoylguanidine 84(trans-2-Phenylcyclopropanecarbonyl)guanidine 714-isopropylcinnamoylguanidine 68 cinnamoylguanidine 576-methoxy-2-naphthoylguanidine 47 2,4-dichlorocinnamolyguanidine 36(4-Chlorocinnamoyl)guanidine 36 5-(N,N-hexamethylene)amiloride 30(4-Bromocinnamoyl)guanidine 29 2,6-dichlorocinnamoylguanidine 275-bromo-2-methoxycinnamoylguanidine 24(5-Phenyl-penta-2,4-dienoyl)guanidine 93-(trifluoromethoxy)cinnamoylguanidine 4 2-t-butylcinnamoylguanidine 4

EXAMPLE 34 Mouse Hepatitis Virus (MHV)

Synthesis and Purification of a Peptide Corresponding to the MHV-A59 EProtein.

A peptide corresponding to the full-length MHV-A59 E protein (sequence:MFNLFLTDTVWYVGQIIFIFAVCLMVTIIVVAFLASIKLCIQLCGLCNTLVLSPSIYLYDRSKQLYKYYNEEMRLPLLEVDDI (SEQ ID NO: 7); accession numberNP_068673) was synthesized manually using FMOC chemistry and solid phasepeptide synthesis The synthesis was done at the Biomolecular ResourceFacility (John Curtin School of Medical Research, ANU, Australia) usinga Symphony^(R) Peptide Synthesiser from Protein Technologies Inc.(Woburn, Mass., USA) according to the manufacturers instructions to giveC-terminal amides, the coupling was done with HBTU andhydroxybenzotriazole in N-methylpyrrolidone. Each of the synthesiscycles used double coupling and a 4-fold excess of the amino acids.Temporary α-N Fmoc-protecting groups were removed using 20% piperidinein DMF.

The crude synthetic peptide was purified using the ProteoPlus™ kit(Qbiogene inc. CA), following manufactures instructions. Briefly, thepeptides were diluted in loading buffer (60 mM Tris-HCl pH 8.3, 6M urea,5% SDS, 10% glycerol, 0.2% Bromophenol blue, ±100 mM β-mercaptoethanol)and run on 4-20% gradient polyacrylamide gels (Gradipore, NSW,Australia) in tris-glycine electrophoresis buffer (25 mM Tris, 250 mMglycine, 0.1% SDS). The peptides were stained with gel code blue(Promega, NSW) and the bands corresponding to the full-length peptidewere excised out of the gel.

The gel slice was transferred to the ProteoPLUS™ tube and filled withtris-glycine electrophoresis buffer. The tubes were emerged intris-glycine electrophoresis buffer and subjected to 100 volts forapproximately 1 hour. The polarity of the electric current was reversedfor 1 minute to increase the amount of protein recovered. The peptideswere harvested and centrifuged at 13,000 rpm for 1 minute. The purifiedpeptides were dried in a Speedvac and the weight of the final productwas used to calculate the yield.

EXAMPLE 35 MHV-E Protein Forms Ion Channels in Planar Lipid Bilayers

Lipid bilayer studies were performed as described elsewhere (Sunstrom,1996; Miller, 1986). A lipid mixture ofpalmitoyl-oleoyl-phosphatidyl-ethanolamine,palmitoyl-oleoyl-phosphatidylserine andpalmitoyl-oleoyl-phosphatidylcholine (5:3:2) (Avanti Polar Lipids,Alabaster, Ala.) was used. The lipid mixture was painted onto anaperture of 150-200 μm in the wall of a 1 ml delrin cup. The apertureseparates two chambers, cis and trans, both containing salt solutions atdifferent concentrations. The cis chamber was connected to ground andthe trans chamber to the input of an Axopatch 200 amplifier. Normallythe cis chamber contained either 500 mM NaCl or 500 mM KCl and the trans50 mM NaCl or 50 mM KCl. The bilayer formation was monitoredelectrically by the amplitude of the current pulse generated by acurrent ramp. The potentials were measured in the trans chamber withrespect to the cis. The synthetic peptide was added to the cis chamberand stirred until channel activity was seen. The currents were filteredat 1000 Hz, digitized at 5000 Hz and stored on magnetic disk.

The MHV E synthetic peptide was dissolved in 2,2,2-trifluorethanol (TFE)at 0.05 mg/ml to 1 mg/ml. 10 μl of this was added to the cis chamber (1ml aqueous volume) of the bilayer apparatus, which was stirred via amagnetic “flea”. Ionic currents, indicating channel activity in thebilayer, were typically detected within 15-30 min. After channels weredetected the holding potential across the bilayer was varied between−100 mV and +100 mV to characterise the size and polarity of currentflow and enable the reversal potential to be determined.

In 14 experiments where the cis chamber contained 500 mM NaCl solutionand the trans chamber contained 50 mM NaCl solution, the averagereversal potential of the channel activity was calculated to be 49±1(SEM) mV. In 11 experiments where the cis chamber contained 500 mM KClsolution and the trans chamber contained 50 mM KCl solution, the averagereversal potential of the channel activity was calculated to be 13±6(SEM) mV. These results indicate that the MHV E protein forms cationselective ion channels that are more selective for Na⁺ than for K⁺ ions.

FIG. 11 shows examples of raw current data for the MHV E ion channel atvarious holding potentials (cis relative to trans) in asymmetrical NaClsolutions (500/50 mM). The graph is a representative plot of averagebilayer current (pA; y-axis) versus holding potential (mV; x-axis).

EXAMPLE 36 Chemical Compounds Inhibit the Ion Channel Activity of theMHV E Protein Synthetic Peptide

To test compounds for their ability to block or otherwise inhibit theion channel formed by MHV E protein, small aliquots of solutionscontaining the compounds were added to the aqueous solutions bathingplanar lipids in which the peptide channel activity had beenreconstituted and the effect of the compound addition on the ioniccurrents was recorded and measured.

Compound stock solutions were typically prepared at 500 mM in DMSO. Thissolution was further diluted to 50 mM, or lower concentration in 50%DMSO/50% methanol and 2 μl of the appropriately diluted compound wasadded to the cis and/or trans chambers to yield the desired finalconcentration.

In the example shown in FIG. 12 below, addition of 100 μMcinnamoylguanidine to the cis chamber greatly reduced current flowthrough the MHV E ion channel.

EXAMPLE 37 Bacterial Bio-Assay for Screening Potential MHV E-Protein IonChannel-Blocking Drugs

MHV E-protein Ion Channel Inhibits Bacterial Cell Growth.

A bio-assay of MHV E-protein function in bacterial cells was developed.A synthetic cDNA fragment encoding MHV E-protein was cloned into theexpression plasmid pPL451, creating a vector in which E proteinexpression is temperature inducible, as described in Example 4.Inhibition of the growth of E.coli cells expressing E protein at 37° C.was observed as an indicator of p7 ion channel function dissipating thenormal Na+ gradient maintained by the bacterial cells.

EXAMPLE 38 Compound Screening Using the Bacterial Bio-Assay for MHV EProtein

The halos of growth around the site of application of particulardrugs—as described in example 14—were scored as described in example 15.

Table 12 lists the scores for inhibition of MHV E protein in thebacterial bio-assay.

TABLE 12 MHV E protein Inhibition Compound (score)4-isopropylcinnamoylguanidine 4.5 3-isopropylcinnamoylguanidinehydrochloride 4.2 4-t-butylcinnamoylguanidine 4.13-(trifluoromethoxy)cinnamoylguanidine 4.1 3-t-butylcinnamoylguanidine4.0 3,4-dichlorocinnamoylguanidine 3.8 2,3-difluorocinnamoylguanidine3.8 2-t-butylcinnamoylguanidine 3.8 3-phenylcinnamoylguanidine 3.72-phenylcinnamoylguanidine 3.4 5-bromo-2-methoxycinnamoylguanidine 3.32-(cyclohex-1-en-1yl)cinnamoylguanidine 3.33-(trifluoromethyl)cinnamoylguanidine 2.93-(cyclohex-1-en-1-yl)cinnamoylguanidine 2.9trans-3-(1-napthyl)acryloylguanidine 2.84-(trifluoromethyl)cinnamoylguanidine 2.8 3-(2-napthyl)acryloylguanidine2.8 2-(trifluoromethyl)cinnamoylguanidine 2.7(4-Phenoxybenzoyl)guanidine 2.4 (3-Bromocinnamoyl)guanidine 2.42,5-dimethylcinnamoylguanidine 2.3 5-bromo-2-fluorocinnamoylguanidine2.1 6-methoxy-2-naphthoylguanidine 1.8 4-phenylbenzoylguanidine 1.8(4-Bromocinnamoyl)guanidine 1.8 1-napthoylguanidine 1.7(5-Phenyl-penta-2,4-dienoyl)guanidine 1.4 (2-Bromocinnamoyl)guanidine1.4 (4-Chlorocinnamoyl)guanidine 1.3 2-methylcinnamoylguanidine 1.22,6-dichlorocinnamoylguanidine 1.2 2,4,6-trimethylcinnamoylguanidine 1.25-(N,N-hexamethylene)amiloride 1.1 cinnamoylguanidine 1.1cinnamoylguanidine hydrochloride 1.1 (a-Methylcinnamoyl)guanidine 1.02,3-dimethylcinnamoylguanidine 1.0 2-cyclohexylcinnamoylguanidine 0.9N-(3-phenylpropanoyl)-N′-phenylguanidine 0.8N,N′-bis(3phenylpropanoyl)-N″-phenylguanidine 0.8(3-Methoxycinnamoyl)guanidine 0.8 (2-Methoxycinnamoyl)guanidine 0.83-fluorocinnamoylguanidine 0.8 2-fluorocinnamoylguanidine 0.82,4-dichlorocinnamolyguanidine 0.8 2-ethylcinnamoylguanidine 0.8(2-Chlorocinnamoyl)guanidine 0.7 (4-Hydroxycinnamoyl)guanidine 0.72-ethoxycinnamoylguanidine 0.7 2-napthoylguanidine 0.6(trans-2-Phenylcyclopropanecarbonyl)guanidine 0.65-(N,N-Dimethyl)amiloride hydrochloride 0.5 5-(4-fluorophenyl)amiloride0.5 3-methylcinnamoylguanidine 0.5 (3-Chlorocinnamoyl)guanidine 0.44-methylcinnamoylguanidine 0.4 4-ethoxycinnamoylguanidine 0.42-(1-napthyl)acetoylguanidine 0.4 3,4-difluorocinnamoylguanidine 0.42-(2-napthyl)acetoylguanidine 0.4 2,3,5,6,-tetramethylcinnamoylguanidine0.4 (4-Methoxycinnamoyl)guanidine 0.33,4-(methylenedioxy)cinnamoylguanidine 0.3 3-ethoxycinnamoylguanidine0.3 4-fluorocinnamoylguanidine 0.2 1-bromo-2-napthoylguanidine 0.25-tert-butylamino-amiloride 0.1 (3-Nitrocinnamoyl)guanidine 0.13,4,5-trimethoxycinnamoylguanidine 0.15-(3′-bromophenyl)penta-2,4-dienoylguanidine 0.1

EXAMPLE 39 MHV Antiviral Assay for Testing Compounds Against Replicationof Mouse Hepatitis Virus (MHV)

To determine the antiviral activity of compounds against MHV replication(strain MHV-A59: ATCC VR-764), an assay measuring reduction in thenumber of plaques formed in monolayers of MHV infected L929 cells (ATCCCCL-a) was developed: First, a virus working stock was prepared byamplification in NCTC clone 1469 cells (ATCC CCL-9.1). This was thenused to infect confluent monolayers of L929 cells grown in 6-well tissueculture plates by exposure to the virus at an MOI of 0.01 pfu/cell or 1pfu/cell for 30 minutes at 37° C. in 5% CO₂. The infective inoculum wasremoved and replaced with fresh medium (DMEM supplemented with 10% horseserum) containing various test concentrations of compounds or theappropriate level of solvent used for the compounds (control). Plateswere subsequently incubated at 37° C. (in 5% CO₂) for 16-24 hours postinfection, after which time culture supernatant was removed and thecells were stained with 0.1% crystal violet solution in 20% ethanol for10 minutes. Plaques were counted in all wells and the percentagereduction in plaque number compared to solvent control was calculated.Measurements were performed in duplicate to quadruplicate wells.

EXAMPLE 40 Effect of Compounds in MHV Antiviral Assay

Table 13 provides the results obtained from this study.

TABLE 13 Percent reduction in Plaque number/# experiments Compound 20 uM10 uM 1 uM 5-(3′-bromophenyl)penta-2,4-dienoyl- N/D 99/2 66/1 guanidine5-bromo-2-methoxycinnamoylguanidine N/D 100/1  66/13-phenylcinnamoylguanidine Toxic 86/2 64/32,3-difluorocinnamoylguanidine Toxic 92/3 64/23-ethoxycinnamoylguanidine 100/1  89/2 58/15-(2′-bromophenyl)penta-2,4-dienoyl- Toxic 100/1  57/1 guanidinecinnamoylguanidine hydrochloride 85/1 72/2 56/1(2-Chlorocinnamoyl)guanidine 95/2 88/3 53/3 cinnamoylguanidine 97/8 88/852/7 (4-Bromocinnamoyl)guanidine Toxic/2 98/3 52/3(2-Bromocinnamoyl)guanidine 91/2 89/3 52/3 (4-Methoxycinnamoyl)guanidine98/4 96/4 51/3 (a-Methylcinnamoyl)guanidine 81/2 75/3 51/23,4-dichlorocinnamoylguanidine 91/2 96/1 50/22-(cyclohex-1-en-1yl)cinnamoylguanidine N/D 97/1 50/13,4-difluorocinnamoylguanidine Toxic 91/2 50/13-t-butylcinnamoylguanidine Toxic 94/3 49/2 2-ethoxycinnamoylguanidine93/2 85/3 48/2 trans-3-Furanacryoylguanidine 70/1 65/1 48/1N-amidino-3-amino-5-hexamethyleneimino- 84/1 52/2 48/16-phenyl-2-pyrazinecarboxamide (2-Nitrocinnamoyl)guanidine 97/1 77/247/1 4-(trifluoromethyl)cinnamoylguanidine 97/3 95/3 46/33,4-(methylenedioxy)cinnamoylguanidine 93/3 82/3 45/35-(N-Methyl-N-isobutyl)amiloride 92/1 85/1 44/2(4-Chlorocinnamoyl)guanidine 97/2 88/2 43/32,4-dichlorocinnamolyguanidine 76/1 73/1 43/1N-(3-phenylpropanoyl)-N′-phenylguanidine 80/1 65/1 43/1(3-Nitrocinnamoyl)guanidine 95/2 77/3 42/3 2-phenylcinnamoylguanidineN/D 100/1  42/1 4-isopropylcinnamoylguanidine 95/3 93/3 41/33-(trifluoromethoxy)cinnamoylguanidine 100/1  90/3 41/23-(trifluoromethyl)cinnamoylguanidine 98/1 83/1 40/1(4-Nitrocinnamoyl)guanidine 97/1 75/3 40/33-(2-napthyl)acryloylguanidine 93/1 93/1 40/1 4-ethoxycinnamoylguanidine96/1 92/1 40/1 2,6-dichlorocinnamoylguanidine 91/1 70/1 40/12,5-dimethylcinnamoylguanidine 95/3 91/3 39/3(3-Bromocinnamoyl)guanidine 95/2 90/3 39/3 (3-Chlorocinnamoyl)guanidine94/1 86/2 39/2 3-methylcinnamoylguanidine 90/1 88/1 39/1(3-Methoxycinnamoyl)guanidine 92/2 87/2 37/3 2-t-butylcinnamoylguanidineN/D 98/2 37/1 [(E)-3-(4-Dimethylaminophenyl)-2- 56/1 45/1 37/1methylacryloyl]guanidine N,N′-bis(1-napthoyl)guanidine 58/1 52/2 35/13-methoxy-HMA 15/1 31/1 35/1 5-tert-butylamino-amiloride 89/4 84/4 34/4trans-3-(1-napthyl)acryloylguanidine 95/2 86/3 34/36-methoxy-2-naphthoylguanidine 88/3 56/3 34/3 2-napthoylguanidine 67/236/2 34/2 2-ethylcinnamoylguanidine 96/1 81/2 34/12,3-dimethylcinnamoylguanidine 95/1 79/2 34/1N″-Cinnamoyl-N,N′-diphenylguanidine 97/1 72/2 34/13-isopropylcinnamoylguanidine N/D 99/2 32/1 hydrochloride(4-Phenoxybenzoyl)guanidine 73/1 65/1 32/1(trans-2-Phenylcyclopropanecarbonyl)- 77/2 64/2 31/2 guanidine3-fluorocinnamoylguanidine 100/1  93/2 31/15-bromo-2-fluorocinnamoylguanidine Toxic 81/2 31/1N,N′-bis-(cinnamoyl)-N″-phenylguanidine 16/1 38/2 31/13-quinolinoylguanidine 27/1 36/2 30/1 2,4,6-trimethylcinnamoylguanidine91/2 61/3 27/2 1-bromo-2-napthoylguanidine 31/1 27/2 27/1N-amidino-3,5-diamino-6-phynyl-2- 53/1 39/2 25/1 pyrazinecarboxamideN-Cinnamoyl-N′,N′-dimethylguanidine 92/2 65/3 24/2(2-Methoxycinnamoyl)guanidine 90/2 85/2 23/22-(2-napthyl)acetoylguanidine 52/1 20/2 23/1 4-phenylcinnamoylguanidine53/1 36/1 21/3 [3-(3-Pyridyl)acryloyl]guanidine 81/2 73/2 21/23,4,5-trimethoxycinnamoylguanidine 84/1 84/1 21/14-methylcinnamoylguanidine 93/1 89/1 20/1 4-fluorocinnamoylguanidine86/1 83/1 20/1 2-methylcinnamoylguanidine 91/1 82/1 20/16-bromo-2-napthoylguanidine 65/1 37/2 19/1 5-(N,N-Dimethyl)amiloridehydrochloride 42/4  7/4 17/4 (5-Phenyl-penta-2,4-dienoyl)guanidine 27/124/1 17/1 2-cyclohexylcinnamoylguanidine 100/1  74/2 16/15-(4-fluorophenyl)amiloride  4/1 25/1 16/1 Benzyoylguanidine 22/1 39/214/1 N-Benzoyl-N′-cinnamoylguanidine  0/1  0/1 14/15-(N,N-hexamethylene)amiloride 84/2 89/1 13/2N-(cinnamoyl)-N′phenylguanidine 83/1 88/1 13/1(4-Hydroxycinnamoyl)guanidine 19/1 15/1 13/12-(trifluoromethyl)cinnamoylguanidine 19/1 15/1 13/1(Quinoline-2-carbonyl)guanidine 86/1 84/1 12/12-(1-napthyl)acetoylguanidine −19/1   02/1 11/12-chloro-6-fluorocinnamoylguanidine 100/1  84/2 9/1N-amidino-3-amino-5-phenyl-6-chloro-2- 20/1 20/2 9/1 pyrazinecarboxamide4-phenylbenzoylguanidine 32/1 24/1  5/1 N,N′-bis(2-napthoyl)guanidine 5/1  3/2  3/1 (Phenylacetyl)guanidine 35/1 22/1  3/11-napthoylguanidine 71/3 62/3  2/3 N,N′-bis(3phenylpropanoyl)-N″-phenyl-67/3 40/4  1/3 guanidine 3-hydroxy-5-hexamethyleneimino-amiloride 16/122/2  1/1 2′4 DichloroBenzamil HCl 12/2  0/3  0/32,3,5,6,-tetramethylcinnamoylguanidine N/D 68/2  0/1 Benzamilhydrochloride  0/1 26/1  0/1 6-Iodoamiloride 28/1 21/1  0/1N,N′-Bis(amidino)napthalene-2,6- 19/1 16/1  0/1 dicarboxamide[(4-Chlorophenoxy-acetyl]guanidine 19/1 16/1  0/1(3-phenylpropanoyl)guanidine 51/1 03/1  0/1 2-fluorocinnamoylguanidine76/1 73/1 6-bromo-2-napthoylguanidine 43/1 (2-Furanacryloyl)guanidine67/2 63/2 −3/2 N-(6-Hydroxy-2-napthoyl)-N′-phenyl- 43/1 39/1 −5/1guanidine Amiloride•HCl 21/1 18/1 −5/13-(trans-hept-1-en-1-yl)cinnamoylguanidine T/1 23(T)/1 −6/13-methoxy-amiloride 60/2 47/3 −7/2 N,N′-Bis(3-phenylpropanoyl)guanidine41/3 30/4 −8/3 3-(cyclohex-1-en-1-yl)cinnamoylguanidine T/1  2/2 −19/1  

EXAMPLE 41 Porcine Respiratory Coronavirus (PRCV)

Antiviral Assay for Testing Compounds Against Replication of PorcineRespiratory Coronavirus (PRCV)

To determine the antiviral activity of compounds against porcinerespiratory coronavirus replication (ATCC VR-2384), an assay measuringreduction in the number of plaques formed in monolayers of PRCV infectedST cells (procine fetal testis cell line, ATCC CRL-1746) was developed:Confluent ST cells in 6 well plates were infected with a quaternarypassage of porcine respiratory virus (PRCV) strain AR310 at threedilutions 10⁻¹, 50⁻¹ and 10⁻² in PBS to provide a range of plaquesnumbers to count. 100 ul of diluted virus was added per well in a volumeof 1 ml of media. Plates were incubated for one hour on a rockingplatform at room temperature to allow virus to adsorb to cells. Theviral supernatant was removed and 2 ml/well of overlay containing 1%Seaplaque agarose in 1×MEM, 5% FCS was added to each well. Compounds tobe tested were added to the overlay mixture by diluting the compoundsfrom frozen stock to a concentration so that the same volume ofcompound/solvent would be added to the overlay for each concentration ofcompound. The volume of compound/solvent never exceeded 0.07% of thevolume of the overlay. The solvent used to dissolve compounds was DMSOand methanol mixed in equal proportions. Compounds were tested foranti-plaque forming activity at four concentrations, 0.1 uM, 1 uM, 10 uMand 20 uM. Either duplicates or quadruplicates were performed at eachconcentration. Controls were performed where the same volume of solventwas added to the overlay. The overlay was allowed to set at room tempfor 20 mins. The plates were then incubated at 37° C. for 2 days. Themonolayers were then fixed and stained overnight at room temperature byadding 1 ml/well of 0.5% methylene blue, 4% formaldehyde. Overlayagarose and stain was then rinsed off to visualize stained and fixedmonolayer

EXAMPLE 42 Effect of Compounds in PRCV Antiviral Assay

Compounds were screened for activity against PRCV replication accordingto the method described in example 29. Table 14 provides EC50 values forsome tested compounds.

TABLE 14 Compound EC50 (uM) 5-(N,N-hexamethylene)amiloride 0.066-methoxy-2-naphthoylguanidine 0.04 cinnamoylguanidine 0.08N-(3-phenylpropanoyl)-N′-phenylguanidine 19 3-methylcinnamoylguanidine1.43 (3-Bromocinnamoyl)guanidine 11.2(trans-2-Phenylcyclopropanecarbonyl)guanidine 17.2trans-3-(1-napthyl)acryloylguanidine 19.1 2-(2-napthyl)acetoylguanidine119.6

EXAMPLE 43 Bovine Coronavirus

Antiviral Assay for Testing Compounds Against Replication of BovineCoronavirus (BCV).

To determine the antiviral activity of compounds against bovinecoronavirus replication (ATCC VR-874), an assay measuring reduction inthe number of plaques formed in monolayers of BCV infected MDBK cells(bovine kidney cell line; ATCC CCL-22) was developed: Confluent MDBKcells in 6 well plates were infected with a secondary passage of BCVwith serially diluted virus diluted to 10⁻³, 5⁻⁵ and 10⁻⁴ in PBS toprovide a range of plaques numbers to count. 100 ul of diluted virus wasadded per well in a volume of 1 ml of media. Plates were incubated forone hr to allow virus to adsorb to cells. The viral supernatant wasremoved and 2 ml/well of overlay containing 1% Seaplaque agarose in1×MEM, 5% FCS was added to each well. Compounds to be tested were addedto the overlay mixture by diluting the compounds from a 0.5M frozenstock to a concentration so that the same volume of compound/solventwould be added to the overlay for each concentration of compound. Thevolume of compound/solvent never exceeded 0.07% of the volume of theoverlay. The solvent used to dissolve compounds was DMSO and methanolmixed in equal proportions. Compounds were tested for anti-plaqueforming activity at four concentrations, 0.1 uM, 1 uM, 10 uM and 20 uM.Quadruplicates were performed at each concentration. Controls wereperformed where the same volume of solvent was added to the overlay. Theoverlay was allowed to set at room temp for 20 mins. The plates werethen incubated at 37° C. for 7 days. The monolayers were then fixed andstained by adding 1 ml/well of 0.5% methylene blue, 4% formaldehyde.

EXAMPLE 44 Effect of Compounds in BCV Antiviral Assay

Compounds were screened for activity against BCV replication accordingto the method described in example 31. Table 15 provides EC50 values forsome tested compounds.

TABLE 15 Compound EC50 uM (3-Bromocinnamoyl)guanidine 33-(trifluoromethyl)cinnamoylguanidine 3 6-methoxy-2-naphthoylguanidine 95-(N,N-hexamethylene)amiloride 9 trans-3-(1-napthyl)acryloylguanidine 13cinnamoylguanidine 42 (5-Phenyl-penta-2,4-dienoyl)guanidine 952-(2-napthyl)acetoylguanidine 99(trans-2-Phenylcyclopropanecarbonyl)guanidine 109N-(3-phenylpropanoyl)-N′-phenylguanidine 156 4-phenylbenzoylguanidine190

EXAMPLE 45 Hepatitis C Virus

Ion Channel Activity of Hepatitis C Virus P7 Protein

Testing of a Synthetic P7 Peptide for channel activity in artificiallipid bilayers

1. A peptide mimicking the protein P7 encoded by the hepatitis C virus(HCV) was synthesised having the following amino acid sequence:

(SEQ ID NO: 8) ALENLVILNAASLAGTHGLVSFLVFFCFAWYLKGRWVPGAVYAFYGMWPLLLLLLALPQRAYA

Lipid bilayer studies were performed as described elsewhere (Miller,1986). A lipid mixture of palmitoyl-oleoyl-phosphatidylethanolamine,palmitoyl-oleoyl-phosphatidylserine andpalmitoyl-oleoyl-phosphatidylcholine (5:3:2) (Avanti Polar Lipids,Alabaster, Ala.) was used. The lipid mixture was painted onto anaperture of 150-200 um in the wall of a 1 ml delrin cup. The apertureseparates two chambers, cis and trans, both containing salt solutions atdifferent concentrations. The cis chamber was connected to ground andthe trans chamber to the input of an Axopatch 200 amplifier. Normallythe cis chamber contained 500 mM KCl and the trans 50 mM KCl. Thebilayer formation was monitored electrically by the amplitude of thecurrent pulse generated by a current ramp. The potentials were measuredin the trans chamber with respect to the cis. The protein was added tothe cis chamber and stirred until channel activity was seen. Thecurrents were filtered at 1000 Hz, digitized at 2000 Hz and stored onmagnetic disk. The P7 peptide was dissolved in 2,2,2-trifluorethanol(TFE) at 10 mg/ml. 10 ul of this was added to the cis chamber of thebilayer which was stirred. Channel activity was seen within 15-20 min.

When the P7 peptide was added to the cis chamber and stirred, channelactivity was recorded. The potential in the trans chamber was −80 mV andthe currents are downwards. The currents reversed at +50 mV close to thepotassium equilibrium potential in these solutions indicating that thechannels were cation-selective. The amplitude of the open-channel peakis 1.7 pA corresponding to a channel conductance of about 14 pS. In mostexperiments, “single channels” had a much larger size, presumablybecause of aggregation of the P7 peptide. The currents reversed at about+40 mV in this experiment. In some experiments the solution in the cischamber was 150 mM KCl and 15 mM KCl in the trans chamber. The P7peptide again produced currents that reversed.

Similar results were obtained when both chambers contained NaCl.Currents recorded in an experiment when the cis chamber contained 500 mMNaCl and the trans chamber 50 mM NaCl. Again the currents reversedbetween +40 and +60 mV, close to the Na⁺ equilibrium potentialindicating that channels were much more permeable to Na⁺ than to K⁺.

The channels formed by the P7 peptide were blocked by5-(N,N-hexamethylene) amiloride (HMA).

Addition of the P7 peptide produced channel activity. Addition of 2 μlof 50 μM HMA to the cis chamber followed by stirring resulted indisappearance of the channel activity. Block of channel activityproduced by the P7 peptide with 100 μM HMA was recorded in 4experiments. In 2 experiments, sodium channels (500/50) were blocked by500 μM HMA

When 10 mM CaCl₂ was added to the cis chamber (K solutions) the reversalpotential of the currents produced by P7 peptide shifted to morenegative potentials indicating a decrease in the P_(K)/P_(Cl) ratio.

When the cis chamber contained 500 mM CaCl₂ and the trans chamber 50 mMCaCl₂, both positive and negative currents were seen at potentialsaround +20 mV and it was not possible to determine a reversal potential.

EXAMPLE 46 Recombinant Expression of HCV p7 Protein

Two cDNA fragments, each encoding the same polypeptide corresponding tothe amino acid sequence of the HCV-1a p7 protein, were synthesisedcommercially by GeneScript. The two cDNAs differed in nucleotidesequence such that in one cDNA (“cDp7.coli”) the codons were optimisedfor expression of the p7 protein in E.coli while in the other cDNA(“cDp7.mam)” codons were biased for expression in mammalian cell lines.cDp7.coli was cloned into the plasmid pPL451 as a BamHI/EcoRI fragmentfor expression in E.coli. cDp7.mam was cloned into vectors (for example,pcDNA3.1 vaccinia virus, pfastBac-1) for expression of p7 in mammaliancell lines.

EXAMPLE 47 Role of p7 in Enhancement of Gat VLP Budding

The budding of virus-like particles (VLP) from cultured HeLa cellsresults from the expression of retroviral Gag proteins in the cells andco-expression of small viral ion channels, such as M2, Vpu and 6K, withthe Gag protein enhances budding. Interestingly, the viral ion channelscan enhance budding of heterologous virus particles. Therefore, toassess budding enhancement by p7 it was co-expressed with the HIV-1 Gagprotein in HeLa cells, and VLP release into the culture medium wasmeasured by Gag ELISA. To achieve this, the plasmids pcDNAp7 (pcDNA3.1=pcDp7.mam as described in Example 20, p7 expressed under controlof the T7 promoter) and pcDNAGag (HIV-1 Gag protein expressed undercontrol of the T7 promoter) were cotransfected into HeLa cells infectedwith the vaccinia virus strain vTF7.3 (expresses T7 RNA polymerase) andculture supernatants were collected for ELISA assay after 16 hoursincubation.

EXAMPLE 48 Assay of the Ability of Compounds to Inhibit p7 Ion ChannelFunctional Activity

The two methods of detecting p7 ion channel functional activity,described in Examples 33-35, were employed to assay the ability ofcompounds to inhibit the p7 channel. In the case of Example 33,compounds were tested for their ability to inhibit p7 channel activityin planar lipid bilayers. In the case of Example 35 compounds weretested for their ability to reduce the number of VLPs released fromcells expressing both p7 and HIV-1 Gag.

EXAMPLE 49

Bacterial Bio-Assay for Screening Potential HCV p7 Protein IonChannel-Blocking Drugs.

HCV p7 Ion Channel Inhibits Bacterial Cell Growth.

A bio-assay of p7 function in bacterial cells was developed. Thep7-encoding synthetic cDNA fragment cDp7.coli was cloned into theexpression plasmid pPL451, creating the vector pPLp7, in which p7expression is temperature inducible, as described in Example 4.Inhibition of the growth of E.coli cells expressing p7 at 37° C. wasobserved as an indicator of p7 ion channel function dissipating thenormal Na+ gradient maintained by the bacterial cells.

EXAMPLE 50 Compound Screening Using the Bacterial Bio-Assay for HCV p7Protein

The halos of growth around the site of application of particulardrugs—as described in example 14—were scored as described in example 15.

Table 16 lists the scores for inhibition of HCV p7 protein in thebacterial bio-assay.

TABLE 16 HCV p7 protein Inhibition (score/# of times Compound tested)2,3-dimethylcinnamoylguanidine 3.88/2 2,4,6-trimethylcinnamoylguanidine3.75/1 5-bromo-2-fluorocinnamoylguanidine 3.73/6(4-Bromocinnamoyl)guanidine 3.47/4 2,5-dimethylcinnamoylguanidine 3.43/43-(trifluoromethyl)cinnamoylguanidine 3.34/34-(trifluoromethyl)cinnamoylguanidine 3.33/56-methoxy-2-naphthoylguanidine 3.33/3 (2-Chlorocinnamoyl)guanidine3.31/6 (4-Chlorocinnamoyl)guanidine 3.16/4 (2-Bromocinnamoyl)guanidine3.00/3 2,6-dichlorocinnamoylguanidine 3.00/3 (3-Bromocinnamoyl)guanidine2.92/3 (3-Chlorocinnamoyl)guanidine 2.75/32-(trifluoromethyl)cinnamoylguanidine 2.63/3 (4-Phenoxybenzoyl)guanidine2.63/1 3,4-dichlorocinnamoylguanidine 2.59/34-isopropylcinnamoylguanidine 2.51/2trans-3-(1-napthyl)acryloylguanidine 2.44/2 4-t-butylcinnamoylguanidine2.42/2 2-t-butylcinnamoylguanidine 2.36/2 2-ethylcinnamoylguanidine2.36/2 4-methylcinnamoylguanidine 2.32/25-bromo-2-methoxycinnamoylguanidine 2.32/23-(trifluoromethoxy)cinnamoylguanidine 2.26/22-cyclohexylcinnamoylguanidine 2.26/2 1-napthoylguanidine 2.25/13-t-butylcinnamoylguanidine 2.23/2 4-phenylbenzoylguanidine 2.19/2(5-Phenyl-penta-2,4-dienoyl)guanidine 2.13/1N-(cinnamoyl)-N′phenylguanidine 2.13/1 3-isopropylcinnamoylguanidinehydrochloride 2.00/1 Benzamil hydrochloride  2.0/1N-(3-phenylpropanoyl)-N′-phenylguanidine  2.0/1N,N′-bis(3phenylpropanoyl)-N″-phenylguanidine  2.0/13-(2-napthyl)acryloylguanidine 1.93/2 5-(N-Methyl-N-isobutyl)amiloride1.88/1 2′4 DichloroBenzamil HCl 1.88/1 5-tert-butylamino-amiloride1.88/1 5-(N-Ethyl-N-isopropyl)amiloride 1.88/1(4-Methoxycinnamoyl)guanidine 1.88/1 4-fluorocinnamoylguanidine 1.86/1(3-Nitrocinnamoyl)guanidine 1.71/1 4-ethoxycinnamoylguanidine 1.63/1(4-Hydroxycinnamoyl)guanidine 1.63/1(trans-2-Phenylcyclopropanecarbonyl)guanidine 1.63/13-ethoxycinnamoylguanidine 1.63/1 2,3,5,6,-tetramethylcinnamoylguanidine1.51/2 4-phenylcinnamoylguanidine  1.5/1 trans-3-Furanacryoylguanidine1.38/1 N-(6-Hydroxy-2-napthoyl)-N′-phenylguanidine 1.38/1(2-Furanacryloyl)guanidine 1.38/13-(cyclohex-1-en-1-yl)cinnamoylguanidine 1.32/2 cinnamoylguanidinehydrochloride 1.32/2 5-(N,N-hexamethylene)amiloride 1.28/42,3-difluorocinnamoylguanidine 1.24/1 2-(1-napthyl)acetoylguanidine1.14/1 (a-Methylcinnamoyl)guanidine 1.14/1 (2-Nitrocinnamoyl)guanidine1.14/1 6-Iodoamiloride 1.13/1 3,4-(methylenedioxy)cinnamoylguanidine1.13/1 2-ethoxycinnamoylguanidine 1.00/1 cinnamoylguanidine 1.00/12-phenylcinnamoylguanidine 1.00/12-(cyclohex-1-en-1yl)cinnamoylguanidine 1.00/1 2-napthoylguanidine 1.0/3 3-phenylcinnamoylguanidine  1.0/1 5-(N,N-Dimethyl)amiloridehydrochloride  1.0/1 5-(4-fluorophenyl)amiloride  1.0/1(3-Methoxycinnamoyl)guanidine  1.0/1 2-fluorocinnamoylguanidine  1.0/15-(3′-bromophenyl)penta-2,4-dienoylguanidine  1.0/1[(4-Chlorophenoxy-acetyl]guanidine  1.0/1 (3-phenylpropanoyl)guanidine 1.0/1 2-chloro-6-fluorocinnamoylguanidine 0.88/13-fluorocinnamoylguanidine 0.86/1 2-methylcinnamoylguanidine 0.75/1(2-Methoxycinnamoyl)guanidine 0.75/1 1-bromo-2-napthoylguanidine 0.75/13,4,5-trimethoxycinnamoylguanidine 0.71/1 3-methylcinnamoylguanidine0.63/1 3-(trans-hept-1-en-1-yl)cinnamoylguanidine 0.50/1 Amiloride•HCl 0.5/2 Phenamil methanesulfonate salt  0.5/12,4-dichlorocinnamolyguanidine 0.38/1 (4-Nitrocinnamoyl)guanidine 0.25/13,4-difluorocinnamoylguanidine 0.13/1 [(E)-3-(4-Dimethylaminophenyl)-2-0.03/4 methylacryloyl]guanidine

EXAMPLE 51 Equine Arteritis Virus (EAV)

Antiviral Assay for Testing Compounds Against Replication of EquineArteritis Virus (EAV).

To determine the antiviral activity of compounds against EAV replication(strain Bucyrus; ATCC VR-796), an assay measuring reduction in thenumber of plaques formed in monolayers of EAV infected BHK-21 cells(ATCC CCL-10) was developed: A virus stock was amplified in RK-13 cells(ATCC CCL-37) and this was then used to infect confluent monolayers ofBHK-21 cells grown in 6-well tissue culture plates by exposure to thevirus at an MOI of 5×10⁻³ pfu/cell for 1 hour at 37° C. 5% CO₂. Theinfective inoculum was removed and the cells were overlayed with a 1%sea plaque overlay (Cambrex Bio Science) in MEM containing 10% FCScontaining and 10, 5 or 1 μM of compounds to be tested or theappropriate level of solvent used for the compounds (control). Plateswere subsequently incubated at 37° C. (in 5% CO₂) for 3 days postinfection, after which time culture supernatant was removed and thecells were stained with 0.1% crystal violet solution in 20% ethanol for10 minutes. Plaques were counted in all wells and the percentagereduction in plaque number compared to solvent control was calculated.Measurements were performed in duplicate to quadruplicate wells.

EXAMPLE 52 Effect of Compounds in EAV Antiviral Assay

Compounds were screened for activity against EAV replication accordingto the method described in example 35. Results expressed as IC50 valuesare shown in Table 17.

TABLE 17 Compound IC50 5-(N,N-hexamethylene)amiloride 7.5 μM(3-Bromocinnamoyl)guanidine  10 μM trans-3-(1-napthyl)acryloylguanidine7.5 μM 2-t-butylcinnamoylguanidine   1 μM2-(cyclohex-1-en-1yl)cinnamoylguanidine  10 μM

EXAMPLE 53 Dengue Flavivirus

Peptide Synthesis of Dengue Virus M Protein

The C-terminal 40 amino acids of the M protein of the Dengue virus type1 strain Singapore S275/90 (Fu et al 1992)(ALRHPGFTVIALFLAHAIGTSITQKGIIFILLMLVTPSMA) (SEQ ID NO: 9) wassynthesised using the Fmoc method. The synthesis was done on a SymphonyPeptide Synthesiser form Protein Technologies Inc (Tucson, Ariz.) asused to give C-terminal amides, the coupling was done with HBTU andhydroxybenzotriazole in N-methylpyrrolidone. Each of the synthesis cycleused double coupling and a 4-fold excess of the amino acids. Temporaryα-N Fmoc-protecting groups were removed using 20% piperidine in DMF.

EXAMPLE 54 Incorporation of Dengue M Virus Protein into Lipid Bilayers

Lipid bilayer studies were performed as described elsewhere (Sunstrom,1996; Miller, 1986). A lipid mixture ofpalmitoyl-oleoyl-phosphatidyl-ethanolamine,palmitoyl-oleoyl-phosphatidylserine andpalmitoyl-oleoyl-phosphatidylcholine (5:3:2) (Avanti Polar Lipids,Alabaster, Ala.) was used. The lipid mixture was painted onto anaperture of 150-200 μm in the wall of a 1 ml delrin cup. The apertureseparates two chambers, cis and trans, both containing salt solutions atdifferent concentrations. The cis chamber was connected to ground andthe trans chamber to the input of an Axopatch 200 amplifier. Normallythe cis chamber contained 500 mM KCl and the trans 50 mM KCl. Thebilayer formation was monitored electrically by the amplitude of thecurrent pulse generated by a current ramp. The potentials were measuredin the trans chamber with respect to the cis. The protein was added tothe cis chamber and stirred until channel activity was seen. Thecurrents were filtered at 1000 Hz, digitized at 5000 Hz and stored onmagnetic disk.

The dengue virus M protein C-terminal peptide (DMVC) was dissolved in2,2,2-trifluorethanol (TFE) at 0.05 mg/ml to 1 mg/ml. 10 μl of this wasadded to the cis chamber of the bilayer which was stirred. Channelactivity was seen within 15-30 min.

EXAMPLE 55 Hexamethylene Amiloride (HMA) to Inhibits Ion ChannelActivity of the Dengue Virus M Protein C-Terminal Peptide

Solutions of 50 mM HMA were prepared by first making a 500 mM solutionin DMSO. This solution was further diluted to 50 mM HMA using 0.1 M HCl.2 μl of the 50 mM HMA was added to the cis chamber after channelactivity was seen. The cis chamber contained 1 ml of solution making thefinal concentration of HMA 100 μM.

EXAMPLE 56 Antiviral Assay for Testing Compounds Against Effects ofDengue Flavivirus Against Cytoproliferation

Compounds were tested at 10, 5, 2.5, 1.25 and 0.625 μM for activityagainst Dengue 1 strain Hawaii using a cytoproliferation assay whichmeasures the effect of dengue virus infection on proliferation ofLLC-MK2, rhesus macaque monkey kidney cells. The LLC-MK2 cells wereinfected with a predetermined amount of virus, titrated such that cellproliferation in infected cultures would be significantly reducedcompared to uninfected controls. The infected cells were then plated at1.5×10³ cells per well in a 96 well plate. Negative controls (no virus,no experimental compound), positive controls (virus, no experimentalcompound), and cytotoxicity controls (experimental compound, no virus)were run with each drug assay. The cultures were allowed to grow for 7days and then Alamar Blue, a fluorescent dye that measures themetabolism of the cultures (red/ox), was added to each culture and thefluorescence value for each culture was measured. The negative controlwithout experimental compound or virus was fixed at 100%. The positivecontrols and the cultures with compound were scored by calculating theiraverage fluorescence as a percentage of the negative control. At leastsix replicate wells were measured for each experimental condition.

EXAMPLE 57 Effect of Compounds in Dengue Antiviral Assay

TABLE 18 Antiviral Drug Percent of Conc. Negative Drug μM Controlcinnamoylguanidine Negative control 0 NA Positive control 0 76.5% 1017.1% 5 38.6% 2.5. 58.3% 1.25 72.6% 0.625 76.6%(2-chlorocinnamoyl)guanidine Negative control 0 NA Positive control 080.3% 10  8.4% 5  7.7% 2.5. 22.7% 1.25 52.5% 0.625 64.3%Trans-3-(1-naphthyl)acryloylguanidine Negative control 0 NA Positivecontrol 0 80.4% 10  6.8% 5 12.4% 2.5. 38.7% 1.25 73.7% 0.625 77.7%N.A.—not applicable

EXAMPLE 58 Positive Correlation Between Bacterial Assay and Anti-ViralAssays EXAMPLE 58.1 Positive Correlation Between Vpu Bacterial Assay andAnti-HIV-1 Data

A correlative study was performed to measure correlation between theactivity scores assigned to compounds tested in the Vpu bacterial assayand the ability of these compounds to inhibit HIV-1 in the anti-viralassay.

EXAMPLE 58.2 Methodology

The p24-antigen data for twelve compounds representing varioussubstituted acylguanidines was compared with the activity scoresobtained for those compounds in the Vpu bacterial assay. The data fromeach assay was initially rank ordered for effectiveness. The rank orderfor the Vpu bacterial assay was determined from all activity scores, thehighest score indicating the greatest effectiveness. The rank order forthe anti-HIV-1 assay was determined based on the overall average valueof p24 antigen measured in culture supernatants at all of the drugconcentrations tested, with the lowest score indicating the greatesteffectiveness. The two rank orders generated were then comparedstatistically by generating the Spearman's Rank correlation coefficient.

EXAMPLE 58.3 Results and Conclusion

The Spearman's correlation coefficient was 0.785 which, by comparisonwith a statistical table of critical values (for n=12), indicates thatthe two rank orders are significantly positively correlated (P<0.01)(Table 19a).

In addition, a different comparison of the Vpu Bacterial assay rankorder with a yes/no score for whether the anti-viral data indicated ap24 reduction of at least one order of magnitude, aligned the ‘yes’group of compounds with the top 6 compounds by the bacterial assay(Table 19b).

These results are indicative that a positive correlation exists betweenbacterial assays and the antiviral assays as performed according to thepresent invention. The bacterial assay may therefore be a useful tool inscreening for compounds that exhibit anti-viral activity.

TABLE 19a Comparison of Rank order of efficacy of 12 substituted acyl-guanidines in the Vpu bacterial assay and anti-HIV assay. Bacterialassay p24 Compound rank order rank order di{circumflex over ( )}2(3-bromocinnamoyl)guanidine 1 1 0 3-(trifluoromethyl)cinnamoylguanidine2 2 0 3-methylcinnamoylguanidine 3 3 0 cinnamoylguanidine 4 4 0trans-3-(1-napthyl)acryloylguanidine 5.5 7 2.256-methoxy-2-naphthoylguanidine 5.5 5 0.25 4-phenylbenzoylguanidine 7 1116 (5-phenyl-penta-2,4-dienoyl)guanidine 8 9 1 N-(3-phenylpropanoyl)-N′-9 12 9 phenylguanidine Hexamethylene amiloride 10 6 162-(2-napthyl)acetoylguanidine 11 10 1 (trans-2- 12 8 16phenylcyclopropanecarbonyl)guanidine Sum di{circumflex over ( )}2 61.5Spearman correlation coefficient 0.785 P value >0.01

TABLE 19b At least 1x log Vpu Bacterial reduction Compounds Bacterialassay rank seen in Ordered by p24 rank order score order p24 assay?(3-bromocinnamoyl)guanidine 4.3 1 yes 3-(trifluoromethyl)cinnamoyl- 3.72 yes guanidine 3-methylcinnamoylguanidine 3.4 3 yes cinnamoylguanidine3.0 4 yes trans-3-(1-napthyl)acryloylguanidine 2.9 5.5 yes6-methoxy-2-naphthoylguanidine 2.9 5.5 yes 4-phenylbenzoylguanidine 2.87 no (5-phenyl-penta-2,4-dienoyl)- 2.6 8 no guanidineN-(3-phenylpropanoyl)-N′- 2.2 9 no phenylguanidine Hexamethyleneamiloride 1.9 10 no 2-(2-napthyl)acetoylguanidine 1.2 11 no (trans-2-0.4 12 no phenylcyclopropanecarbonyl)- guanidine

EXAMPLE 58.4 Correlation Between Percent Inhibition of MHV PlaqueFormation and MHV-E Bacterial Bio-Assay Score

A positive correlation was seen between the activity scores assigned tocompounds when tested in the Mouse Hepatitis Virus E-protein bacterialbio-assay and the percent inhibition exhibited by these compounds in theMouse Hepatitis Virus plaque assay.

EXAMPLE 58.5 Method

MHV plaque reduction activity data for 96 compounds screened were sortedfrom greatest to least percent plaque reduction and rank orders wereassigned to the list of compounds. This was performed for the datagenerated by exposure to both 10 μM and 1 μM concentrations of thecompounds, giving rise to two rank order lists.

Similarly, a rank order list was generated for the MHVE bacterialbioassay scores for the same 96 compounds. Where one or more compoundshad the same score, the rank values for that group were averaged.

Spearman's statistical test for [as described in “MathematicalStatistics with Applications” (2^(nd) edn): Mendenhall, W., Scheaffer, RL., & Wackerly, D D. Duxbury Press, Boston Mass.,—1981] was used tocompare rank orders. Briefly, this involved calculating the Sum ofsquares (SS) of the differences between rank values for each compound,and then generating the Spearman's Rank Correlation coefficient (Rs)according to the formula: Rs=1−(6·SS/n(n²−1)), where n is the number ofcompounds ranked (96 in this case). Rs is then compared to a Table ofcritical values to determine statistical significance (P values).

EXAMPLE 58.6 Summary of Results

This table summarises the Rs and P values generated as a result of theindicated pairwise comparisons between rank orders.

TABLE 20 Comparison Rs P +ive correlation Plaque at 10 μM Plaque at 1 μM0.689 >99.5 Yes Bacterial Plaque at 10 μM 0.444 >99 Yes Plaque at 1 μM0.406 >98.5 Yes Randomised order −0.382 n/s No

EXAMPLE 58.7 Conclusions

The rank order comparison of 96 compounds assayed in the bacterialbio-assay and the antiviral assay show that MHVE bacterial assay rankorder for the compounds tested is significantly positively correlatedwith the rank orders generated by the MHV plaque reduction assay. Thesignificant correlation between the assays is highly indicative thateither assay may be utilised to identify compounds that may be useful.The bacterial assay may thereby be a useful tool in screening forcompounds that exhibit anti-viral activity.

EXAMPLE 58.8 Correlation Between Percent Inhibition of 229E PlaqueFormation and 229E-E Bacterial Bio-Assay Score

A positive correlation was seen between the activity scores assigned tocompounds when tested in the Human Coronavirus 229E E-protein bacterialbio-assay and the percent inhibition exhibited by these compounds in theHuman Coronavirus 229E plaque assay.

EXAMPLE 58.9 Method

229E plaque reduction activity data for 97 compounds screened against2.5 μM compound concentration were sorted from greatest to least percentplaque reduction and rank orders were assigned to the list of compounds.Similarly, a rank order list was generated for the 229E E bacterialbioassay scores for the same 97 compounds. Where one or more compoundshad the same score, the rank values for that group were averaged.

Spearman's statistical test for [as described in “MathematicalStatistics with Applications” (2^(nd) edn): Mendenhall, W., Scheaffer, RL., & Wackerly, D D. Duxbury Press, Boston Mass.—1981] was used tocompare rank orders. Briefly, this involves calculating the Sum ofsquares (SS) of the differences between rank values for each compound,and then generating the Spearman's Rank Correlation coefficient (Rs)according to the formula: Rs=1−(6·SS/n(n²−1)), where n is the number ofcompounds ranked (97 in this case). Rs is then compared to a Table ofcritical values to determine statistical significance (P values).

EXAMPLE 58.9.1 Summary of Results and Conclusions

This table summarises the Rs and P values generated as a result of theindicated pairwise comparisons between rank orders.

TABLE 21 Comparison Rs P +ive correlation Plaque at 2.5 μM Bacterial0.584 >99.5 Yes randomised −0.382 n/s No

The results above indicate that the 229E bacterial assay rank order forthe compounds tested is significantly positively correlated with therank orders generated by the 229E plaque reduction assay. This resultcombined with that shown in Examples 49.1 and 49.4, provides strongevidence that either assay may be utilised to identify compounds thatmay be useful. The bacterial assay may thereby be a useful tool inscreening for compounds that exhibit anti-viral activity.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

BIBLIOGRAPHY

-   Barry, M., Mulcahy, F. and Back, D-J., Br J Clin Pharmacol, 45: 221    (1998)-   Deeks, S. G., West J Med, 168:133 (1998)-   Miles, S. A., J Acquir Immune Defic Syndr Hum Retrovirol, 16 Suppl    1: S36 (1997)-   Miles, S. A., J. Acquir Immune Defic Syndr Hum Retrovirol, 16 Suppl    1: S I (1998)-   Moyle, G. J., Gazzard, B. G., Cooper, D. A. and Gatell, J., Drugs,    55:383 (1998)-   Rachlis, A. R. and Zarowny, D. P., Cmaj, 158:496 (1998)-   Vella, S., Fragola, V. and Palmisano, L., J Biol Regul Homeost    Agents, 11:60 (1997)-   Volberding, P. A. and Deeks, S. G., Jama, 279:1343 (1998)-   Volberding, P. A., Hosp Pract (Off Ed), 33:81-4, 87-90, 95-6 passim.    (1998)-   Miller, R. H. and Sarver, N., Nat Med, 3:389 (1997)-   Mitsuya, H., Enzyme Inhibition, 6:1 (1992)-   Moore, J. P., Science, 276:51 (1997)-   Thomas, M. and Brady, L., Trends Biotechnol., 15:167 (1997)-   Balliet, J. W., Kolson, D. L., Eiger, G., Kim, F. M., McGann, K. A.,    Srinivasan, A. and Collman, R., Virology, 200:623 (1994)-   Westervelt, P., Henkel, T., Trowbridge, D. B., Orenstein, J.,    Heuser, J., Gendelman, H. E. and Ratner, L., J Virol, 66:3925 (1992)-   Ewart, G. D., Sutherland, T., Gage, P. W. and Cox, G. B., J Virol,    70:7108 (1996)-   Schubert, U., Henklein, P., Boldyreff, B., Wingender, E.,    Strebel, K. and Porstmann, T. J Mol Biol, 236:16 (1994)-   Friborg, J., Ladha, A., Gottlinger, H., Haseltine, W. A. and    Cohen, E. A., Journal of Acquired Immune Deficiency Syndromes &    Human Retrovirology, 8:10 (1995)-   Fu, J., Tan, B. H., Yap, E. H., Chan, Y. C. and Tan, Y. H. (1992)    Full-length cDNA sequence of dengue type 1 virus (Singapore strain    S275/90). Virology 188 (2), 953-958-   Love, C. A., Lilley, P. E. and Dixon, N. E., Escherichia coli. Gene,    in press. (1996)-   Yamato, I., Kotani, M., Oka, Y. and Anraku, Y., Escherichia coli.    Journal of Biological Chemistry, 269:5729 (1994)-   Rosen, B. R., ATP-coupled solute transport systems. Escherichia coli    and Salmonella typhimurium: Cellular and molecular biology 1: (1987)    Editor: Neidhardt, F. C. American Society for Microbiology.-   Piller, S. C., Ewart, G. D., Premkumar, A., Cox, G. B. and Gage, P.    W., Proceedings of the National Academy of Sciences of the United    States of America, 93:111 (1996) Lu, Y. A., Clavijo, P., Galantino,    M., Shen, Z. Y., Liu, W. and Tam, J. P., Molecular Immunology,    28:623 (1991)-   Harlow, E. and Lane, D., (1988) Antibodies: A laboratory manual.    (ed). Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,    ‘Vol:’.-   Varadhachary, A. and Maloney. P-C., Molecular Microbiology, 4:1407    (1990)-44-   New, R. C. C., (1990). Liposomes: A practical approach. The    Practical Approach Series.-   Kuhn R J, Zhang W, Rossmann M G, Pletnev S V, Corver J, Lenches E,    Jones C T, Mukhopadhyay S, Chipman P R, Strauss E G, Baker T S,    Strauss J H. (2002). Structure of dengue virus: implications for    flavivirus organization, maturation, and fusion. Cell. 2002 Mar. 8;    108(5):717-25.-   Rickwood, D., Harries, B. D. (eds). IRL Press, Oxford.-   Miller, C., (1986) Ion channel reconstitution. (ed). Plenum Press,    New York and London.-   Fear, W. R., Kesson, A. M., Naif, H., Lynch, G. W. and    Cunningham, A. L., J. Virol, 72:1334 (1998)-   Kelly, M. D., Naif, H., Adams, S. L., Cunningham, A. L. and    Lloyd, A. R., J. Immunol, 160:3091 (1998)-   New, R. C. C. (ed.), Liposomes: a practical approach. IRL Press,    Oxford (1990) Grice, A. L., Kerr, I. D. and Sansom, M. S., FEBSLett,    405(3):299-304 (1997) Moore, P. B., Zhong, Q., Husslein, T. and    Klein, M. L., FEBS Lett, 431(2):143-148 (1998) Schubert, U., Bour,    S., Ferrermontiel, A. V., Montal, M., Maldarelli, F. and Strebel,    K., Journal of Virology, 70(2):809-819 (1996a)-   Sunstrom N A, Premkumar L S, Premkumar A, Ewart G, Cox G B, Gage P W    (1996), Ion channels formed by NB, an influenza B virus protein. J    Membr Biol. 1996 March; 150(2): 127-32-   Willbold, D., Hofftnann, S. and Rosch, P., Eur J Biochem,    245(3):581-8 (1997) Wray, V., Kinder, R., Federau, T., Henklein, P.    Bechinger, B. and Schubert, U., Biochemistry, 38(16):5272-82 (1999)

The invention claimed is:
 1. A pharmaceutical composition comprising acompound selected from the group consisting of: 4-phenylbenzoylguanidine

N-(3-phenylpropanoyl)-N′-phenyl-guanidine

N,N′-bis(3-phenylpropanoyl)guanidine

N-(6-Hydroxy-2-naphthoyl)-N′-phenyl-guanidine

N-Benzoyl-N′-cinnamoylguanidine

(5-Phenyl-penta-2,4-dienoyl)guanidine

4-phenylcinnamoylguanidine

5-(3′-bromophenyl)penta-2,4-dienoylguanidine

3-phenylcinnamoylguanidine

5-(2′-bromophenyl)penta-2,4-dienoylguanidine

2-cyclohexylcinnamoylguanidine

2-phenylcinnamoylguanidine

2-(cyclohex-1-en-1yl)cinnamoylguanidine

and a pharmaceutically acceptable salt, wherein the composition hasanti-viral activity.
 2. The compound according to claim 1 wherein thecompound is selected from: 2-phenylcinnamoylguanidine,3-phenylcinnamoylguanidine, 4-phenyl cinnamoylguanidine,5-(2′-bromophenyl)penta-2,4-dienoylguanidine,5-(3′-bromophenyl)penta-2,4-dienoylguanidine,N-(6-Hydroxy-2-naphthoyl)-N′-phenylguanidine, andN,N′-Bis(3-phenylpropanoyl)guanidine, or a pharmaceutically acceptablesalt thereof.
 3. A method for the therapeutic or prophylactic treatmentof a subject infected with or exposed to a virus, comprisingadministering to the subject a compound according to claim 1, whereinsaid virus is a Lentivirus, Human Immunodeficiency Virus (HIV), aCoronavirus, the Hepatitis C virus or Equine Arteritis virus.
 4. Thepharmaceutical composition according to claim 1 wherein the compound isselected from: N-(6-Hydroxy-2-naphthoyl)-N′-phenyl-guanidine,5-(3′-bromophenyl)penta-2,4-dienoylguanidine,5-(2′-bromophenyl)penta-2,4-dienoylguanidine and2-phenylcinnamoylguanidine.
 5. The pharmaceutical composition accordingto claim 1, wherein the composition has activity against Lentivirus,Human Immunodeficiency Virus (HIV), a Coronavirus, Hepatitis C virus orEquine Arteritis virus.